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

Summary

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.

Abstract

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.

Introduction

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 growth^1,2,3. 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 scar^1,3. Despite this, the capacity to regenerate long axons to distant targets and to form appropriate functional synapses remains severely limited^4,5,6,7.

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 µm/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.

Protocol

All procedures detailed below were approved by McGill University'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

1. 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).
2. 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.
3. Coat the dishes or coverslips with 0.5-1 mL of 100 µg/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.
4. 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.
5. 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.
6. 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 .
7. To fill the single population device with medium, point the pipette towards the channels and add 50 µL of complete cell medium supplemented with serum-free B-27 (volume ratio 1:50) and 500 µg/mL penicillin/streptomycin/glutamine (collectively called NBM) to the right upper well, and then add another 50 µL to the well lying diagonally to it. Do this for all devices, making sure that the medium flows between wells. Next, add 50 µL of medium to the remaining 2 wells. See Figure 2a .
   1. To fill the multiple population device with medium, point the pipette towards the channels and add 30 µL 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 µL of medium to the remaining 4 wells.
8. Place the devices in a bigger plate with an open dish with autoclaved water (wet chamber) and place in the incubator (37 °C, 5% CO₂ and 95% humidity) for 1-2 h while preparing the cell culture. See Figure 2b .

2. Plating Neurons in Microfluidic Systems

1. Following the protocol outlined in Ref.⁸, obtain dissociated hippocampal or cortical neurons from Sprague Dawley rat embryos (either gender).
2. 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.
3. Remove the medium from the microfluidic devices without emptying the wells. Leave approximately 5 µL in each.
4. To plate cells in the single population device, add 50 µL of NBM to the lower right well. At this point the medium flows by itself to fill the other lower well. Add 20 µL of the concentrate cell solution into the top right well of the microfluidic device, as indicated in Figure 1b .
   1. To plate cells in the multiple population device add 20 µL of the concentrate cell solution into each of the right wells in Figure 6a.
5. 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.
6. Check in the microscope if there are enough cells in the chambers. If more are needed, repeat steps 2.4 and 2.5.
7. Add 50 µL of NBM to the 2 top wells of the single population device and 20 µL 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.
8. Maintain the cells at 37 °C, 5% CO₂ and 95% humidity.

3. Maintaining the Neuronal Cultures

1. Remove NBM (roughly 30 µL 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).
2. 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.
3. 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.

4. Removal of Microfluidic Devices

1. 1 - 2 d before removal of microfluidic devices, add 2 mL of NBM prewarmed to 37 °C to each sample dish, flooding the chambers, and maintain the devices in the incubator.
2. 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.
3. Every 2-3 days, replace half the NBM until the sample is used for experiments.
4. 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.

5. Preparing PDL-coated Beads

1. Add 2 x 50 µL drops of either 4, 10 or 20 µm polystyrene beads diluted in water (1:500) to 1 mL of PDL (100 µg/mL). Leave for at least 2 h at room temperature.
   Note: Protocol can be paused here and resumed the following day.
2. Centrifuge the solution at 8,820 x g for 1 min. Carefully remove supernatant without disturbing the beads accumulated at bottom of container.
3. Wash the beads twice with 1 mL of sterile 10 mM HEPES pH 8.4 solution.
4. Resuspend the PDL-coated beads in 200 mL of 10 mM HEPES pH 8.4 solution.

6. Preparing Micropipettes

1. 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 µm. Before pulling, ensure the glass tubes are clean.
2. 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.

7. PDL-bead Adhesion to Neurons

1. Add 40-60 µL 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).
2. Return the sample to the incubator for 1 h to promote the formation of synaptic contacts^8,9.
3. After the incubation, remove any un-adhered beads by gently washing the culture with pre-warmed NBM.

8. Preparing Physiological Saline Solution (for Room Temperature Experiments)

1. Prepare physiological saline solution by combining the ingredients listed in references^7,8. This is to regulate the cell environment outside the incubator.
2. Verify osmolarity and pH levels as indicated in references^7,8.
3. Continuously infuse solution with O₂ to minimize pH fluctuations while conducting experiments.
4. Heat to room temperature.
5. 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).
   1. 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.

9. Bead Micromanipulation

1. 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).
2. During experiments, continuously perfuse cells with the physiological saline solution prepared in step 8 at a rate of 0.5-1 mL/min.
3. 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.
4. Apply negative pressure with the 1 mL syringe connected to the pipette to pick up the bead. Maintain negative pressure throughout the experiment.

10. Pulling Neurites

1. 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.
2. Pull the PDL-bead-neuron complex by slowly (~0.5 µm/min) moving either the micromanipulator or the sample stage by 1 µm and pause for 5 min to allow neurite initiation.
3. Repeat step 10.2 twice.
   Note: The first 3 µm have to be pulled very slowly to guarantee experimental success, which occurs over 95% of the time.
4. Pull the PDL-bead-neuron complex by slowly (~0.5 µm/min) moving either the micromanipulator or the sample stage by 2 µm and pause for 5 min to allow neurite elongation.
5. After successful initiation and neurite extension for the first 5 µm, pull the neurite at 20 µm/min over millimeter-scale distances.
   Note: Pulling can be performed continuously or in steps and at varying rates. See Figure 5b-5c .

11. Connecting Neurons

1. 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.
2. 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 µm form the first bead. Use the second PDL-bead to push the new neurite filament towards the target cell.
3. 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.
4. During this time, use perfusion to slowly change the medium of the sample from physiological saline to pre-warmed, CO₂-equilibrated NBM.
5. 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 .
6. Gently remove saline solution and replace with NBM (~2 mL).
7. Carefully place the sample back in the incubator to strengthen the neuronal connection for future experiments. This connection is stable for >24 h⁷.

12. Verifying the Functionality of the New Connection via Whole-cell Paired Patch Clamp Recordings

1. Follow references^7,18,19. to assemble an electrophysiology set-up.
2. Follow references⁷. to prepare the pre- and postsynaptic electrodes.
3. Gather patch clamp data, again following references^18,19.
4. Compare the results to naturally occurring signals⁷ to determine the connection type.

Results

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...

Discussion

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 labs^4,13. 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 ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Co-culture devices	Ananda Devices		Commercially available at http://www.anandadevices.com
Neuro devices	Ananda Devices		Commercially available at http://www.anandadevices.com
No. 1 Glass Coverslip 25 mm Round	Warner Instruments	64-0705
35 mm Glass Bottom Dishes #0, Uncoated, Gamma-Irradiated	MatTex Incorporation	P35G-0-20-C
35 mm cell culture dish, Non-Pyrogenic, Sterile	Corning Inc	430165
95 mm x 15 mm Petri Dish, Slippable Lid, Sterile Polystyrene	Fisherbrand	FB0875714G
50 mL Centrifuge tubes with printed graduations and flat caps	VWR	89039-656
15 mL Polypropylene Conical Tube, 17 x 120 mm style, Non Pyrogenic, Sterile	Falcon	352097
Neurobasal Medium	Life Technologies	21103-049	Extracellular solution
B-27 Supplement (50X), serum free	B-27 Supplement (50X), serum free	17504044	Extracellular solution
Pennicilin, Streptomyocin, Glutamine	Thermo Fisher Scientific	11995-065	Extracellular solution
200 μ L Pipettors	VWR	89079-458
2 - 20 μL Pipettors	Aerosol Resistant Tips	2149P
BD Falcon 3mL Transfer Pipettes [Non-sterile]	BD Falcon	357524
Glucose	Gibco	15023-021	Extracellular solution
HEPES	Sigma	7365-45-9	Extracellular solution/Beads
NaCl	Sigma-Aldrich	7647-14-5	Extracellular solution
KCl	Sigma-Aldrich	7447-40-7	Extracellular solution
CaCl2	Sigma-Aldrich	10043-52-4	Extracellular solution
MgCl2	Sigma-Aldrich	7786-30-3	Extracellular solution
#5 Dumont Dumostar Tweezers 11 cm	World Precision Instruments	500233
Dissection tools	Braun, Aesculap
Poly-D-lysine Hydrobromide	Sigma-Aldrich	P6407
Micro particles based on polystyrene, 10 μm	Sigma-Aldrich	72986
Borosilicate tubes	King Precision Glass, Inc.	14696-2
Horizontal Pipette Puller	Sutter Instruments	Brown-Flaming P-97
Micromanipulators, PCS-5000 Series	SD Instruments	MC7600R
1 mL Syringe	BD Luer-Lok	309628
Inverted Microscope	Olympus	IX71
Objective	Olympus	UIS2, LUCPLFLN 40X
CCD Camera	Photometrics	Cascade II: 512
Leibovitz's (1x) L-15 Medium	Life Technologies	11415-064	Rat Dissection
Typsin-EDTA (0.05%), Phenol red	Life Technologies	25300054	Rat Dissection
DMEM (1x) Dulbecco's Modified Eagle Medium [+4.5 g/L D-Glucose, + L-Glutamine, + 110 mg/L Sodium Pyruvate]	Life Technologies	11995-065	Rat Dissection
HBSS (1x) Hank's Balanced Salt Solution [- Calcium Chloride, - Magnesium Chloride, - Magnesium Sulfate]	Life Technologies	14170-112	Rat Dissection