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Optogenetic Stimulation and Recording of Primary Cultured Neurons with Spatiotemporal Control
对原代培养神经元进行时空控制的光遗传学刺激和记录   

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Abstract

We studied a network of cortical neurons in culture and developed an innovative optical device to stimulate optogenetically a large neuronal population with both spatial and temporal precision. We first describe how to culture primary neurons expressing channelrhodopsin. We then detail the optogenetic setup based on the workings of a fast Digital Light Processing (DLP) projector. The setup is able to stimulate tens to hundreds neurons with independent trains of light pulses that evoked action potentials with high temporal resolution. During photostimulation, network activity was monitored using patch-clamp recordings of up to 4 neurons. The experiment is ideally suited to study recurrent network dynamics or biological processes such as plasticity or homeostasis in a network of neurons when a sub-population is activated by distinct stimuli whose characteristics (correlation, rate, and, size) were finely controlled.

Keywords: Primary culture of neurons(神经元的原代培养), Optogenetics(光遗传学), Patterned optical stimulation(模式化光学刺激)

Background

Optogenetics provide a mean to control neuronal activity with millisecond precision. However, neurons are often activated simultaneously either by flashes of light that activate the whole population synchronously or by a light whose intensity is temporally modulated over the whole field of view (Boyden et al., 2005). Yet, several methods exist to modulate the light spatially and have been used to uncage glutamate (Nawrot et al., 2009) or activate channelrhodopsin (ChR2) expressing neurons (Guo et al., 2009) (for review of available methods to stimulate neurons with both spatial and temporal resolution see Anselmi et al., 2015).

To gain spatial control of the stimulation, a first possibility is to use a laser and move its beam quickly over different positions. For example, uncaging glutamate at different dendritic locations has been achieved by deflecting a laser beam with acousto-optic deflectors (Shoham et al., 2005). This strategy is likely viable only if we modulate the light intensity sufficiently slowly over a limited area. Alternatively, a spatial pattern of light can be achieved using phase or intensity light modulators. Holographic technique based on phase modulation permits to obtain an image in three dimensions with a good spatial precision but patterns can be displayed at a rate of only 100 Hz (Papagiakoumou et al., 2010). If a two dimensional pattern is sufficient, intensity modulation can simply be obtained by placing a projector or an array of LEDs in the conjugated plane of the sample (Farah et al., 2007; Guo et al., 2009). This technique has the advantages of being easy to implement, can target many regions of interest simultaneously and has the fastest temporal resolution.

Here we took advantage of a fast video projector based on the workings of a Digital Micromirror Device (DMD). A LED light source is split by an array of micromirrors that can be controlled with sub millisecond precision in order to display any arbitrary pattern of light (Barral and Reyes, 2016). An image of the projector is focalized to the sample plane via a pair of lenses and the microscope objective. The DMD technology offers an unprecedented temporal precision that enables to display patterns at 1.44 kHz and even faster DMDs are now available. In our settings, the resulting pixel size (2.2 x 1.1 µm) was sufficiently small to stimulate single neurons.

To activate a single neuron, we selected a region of interest of ~30 x 30 µm, centered at the soma of the neuron of interest and sent a 5 msec pulse of light. By designing patterns that are projected onto the sample, we could target independently and simultaneously a large number of neurons (10 to 100 neurons). Stimulated neurons were both excitatory and inhibitory (expression of ChR2 under the Synapsin promoter) and were activated by Poisson spike trains. The rate and correlation of the spike stimuli were controlled by the experimenter (see Barral and Reyes, 2016). By recording from neurons that expressed ChR2, we verified that stimulated neurons responded faithfully to the light pulses. We then recorded concurrently the membrane potentials of up to 4 neurons in cell-attached and in whole-cell configurations to isolate the spiking activity and the postsynaptic inputs, respectively.

Materials and Reagents

  1. For the neuronal culture
    1. Round coverslips, 25 mm diameter, German glass (Electron Microscopy Sciences, catalog number: 72196-25 )
    2. Siliconized low-retention microcentrifuge tubes (1.5 ml) (Fisher Scientific, catalog number: 02-681-331 )
    3. Low-retention pipet tips 200 µl (Fisher Scientific, catalog number: 02-717-165 )
    4. Low-retention pipet tips 1,000 µl (Fisher Scientific, catalog number: 02-717-166 )
    5. Disposable Petri dishes (35 x 10 mm) (Corning, Falcon®, catalog number: 351008 )
    6. Stericup-GP sterile vacuum filtration system, 0.22 µm, polyethersulfone (EMD Millipore, catalog number: SCGPU05RE )
    7. Syringe filters; MCE membrane; pore size: 0.22 µm (EMD Millipore, catalog number: SLGS033SS )
    8. Sterile transfer pipets (Fisher Scientific, catalog number: 13-711-20 )
    9. Conical sterile polypropylene centrifuge tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339650 )
    10. Postnatal (P0-P1) mice
    11. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 320331 )
    12. Nitric acid (Sigma-Aldrich, catalog number: 258121 )
    13. 70% ethanol
    14. Poly-L-lysine hydrobromide–mol wt 70,000-150,000 (Sigma-Aldrich, catalog number: P1274 )
    15. Sodium tetraborate decahydrate ≥ 99.5% (Sigma-Aldrich, catalog number: B9876-500G )
    16. Boric acid (cell culture tested) (Sigma-Aldrich, catalog number: B9645-500G )
    17. HBSS (10x), no calcium, no magnesium, no phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 14185052 )
    18. Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
    19. HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080 )
    20. Agar (Sigma-Aldrich, catalog number: A1296-100G )
    21. Sodium bicarbonate (NaHCO3) (Fisher Scientific, catalog number: S233 )
    22. Sodium pyruvate solution (Sigma-Aldrich, catalog number: S8636-100ML )
    23. Sodium hydroxide (NaOH) (Fisher Scientific, catalog number: S318 )
    24. L-cysteine hydrochloride monohydrate (Sigma-Aldrich, catalog number: C7880-500MG )
    25. DNase I grade II, from bovine pancreas–100 mg (Roche Diagnostics, catalog number: 10104159001 )
    26. Papain from Carica papaya–10 ml 100 mg (Roche Diagnostics, catalog number: 10108014001 )
    27. Calcium chloride (CaCl2) (Fisher Scientific, catalog number: C79 )
    28. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
    29. Trypsin inhibitor from chicken egg white (Sigma-Aldrich, catalog number: T9253-500MG )
    30. Albumin from bovine serum (powder, suitable for cell culture, = 96%) (Sigma-Aldrich, catalog number: A9418-5G )
    31. Trypan blue solution (0.4%, liquid, sterile-filtered, suitable for cell culture) (Sigma-Aldrich, catalog number: T8154-20ML )
    32. Neurobasal medium (1x) (Thermo Fisher Scientific, GibcoTM, catalog number: 21103049 )
    33. B-27 serum-free supplement containing vitamin A (50x), liquid (Thermo Fisher Scientific, GibcoTM, catalog number: 17504044 )
    34. GlutaMAXTM supplement (Thermo Fisher Scientific, GibcoTM, catalog number: 35050061 )
    35. AAV virus for ChR2 expression (University of North Carolina Vector Core Services, AAV2-hSyn-hChR2(H134R)-mCherry)
    36. Sodium chloride (NaCl) (Fisher Scientific, catalog number: BP358 )
    37. D-glucose (Fisher Scientific, catalog number: BP350 )
    38. Potassium chloride (KCl) (Fisher Scientific, catalog number: P333 )
    39. Sodium phosphate monobasic (NaH2PO4) (Fisher Scientific, catalog number: S369 )
    40. K-gluconate (Sigma-Aldrich, catalog number: P1847 )
    41. Phosphocreatine (Sigma-Aldrich, catalog number: P1937 )
    42. ATP-Mg (Sigma-Aldrich, catalog number: A9187 )
    43. GTP (Sigma-Aldrich, catalog number: G8877 )
    44. Poly-L-Lysine (PLL solution) (see Recipes)
    45. Dissection solution (see Recipes)
    46. Papain solution (see Recipes)
    47. DNase/L-cysteine solution (see Recipes)
    48. DNase/Mg solution (see Recipes)
    49. Trypsin inhibitor solution (see Recipes)
    50. Culture medium (NB/B27 medium) (see Recipes)

  2. For electrophysiology recordings
    1. Borosilicate glass capillaries (1.5 OD) (World Precision Instruments, catalog number: 1B150F-4 )
    2. Artificial cerebrospinal fluid (aCSF, see Recipes)
    3. Intracellular solution (see Recipes)

Equipment

  1. For the neuronal culture
    1. Tissue culture hood
    2. P1000 pipet
    3. P200 pipet
    4. Cell culture incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: FormaTM Series II 3110 , catalog number: 3110)
    5. Vibratome slicer (Leica Biosystems, model: Leica VT1200 S )
    6. Dumont #5 forceps (Fine Science Tools, catalog number: 11251-30 )
    7. Dumont #7 forceps (Fine Science Tools, catalog number: 11271-30 )
    8. Extra fine bonn scissors (straight) (Fine Science Tools, catalog number: 14084-08 )

  2. For the electrophysiology setup
    1. Upright water immersion microscope with fluorescence (Olympus, model: BX51 )
    2. Fluorescence filter set for mCherry (TRITC, Chroma Technology, model: 41002c )
    3. CCD camera for fluorescence imaging (Hamamatsu Photonics, model: C8484 )
    4. CCD camera for IR imaging (Olympus, model: OLY-150 IR )
    5. Micromanipulator (Luigs & Neumann, model: SM6 )
    6. Patch-clamp amplifiers (Dagan, model: BVC-700A )
    7. 18-bit interface card (National Instruments, model: PCI-6289 )
    8. Flaming/Brown micropipette puller (Sutter Instrument, model: P-97 )
    9. Hemacytometer (Hausser, catalog number: 3110 )

  3. For the optogenetic setup
    1. DLP projector (Texas Instruments, model: DLP® LightCrafterTM Evaluation Module )
    2. Aluminum Breadboard, 150 x 300 x 12.7 mm, double density, M6 thread (Thorlabs, model: MB1530/M )
    3. Ø1" Achromatic Doublet, SM1-Threaded Mount, f = 35 mm, ARC: 400-700 nm (Thorlabs, model: AC254-035-A-ML )
    4. Ø2" Achromatic Doublet, SM2-Threaded Mount, f = 200 mm, ARC: 400-700 nm (Thorlabs, model: AC508-200-A-ML )
    5. Ø2" (Ø50.8 mm) Protected Silver Mirror, 0.47" (12.0 mm) Thick (Thorlabs, model: PF20-03-P01 )
    6. Kinematic Mount for Ø2" Optics (Thorlabs, model: KM200 )
    7. U-DP; Dual port intermediate tube (Olympus, model: U-IT140 )
    8. U-EPA2; Eyepoint adjuster, BX2, Raises eyepoint 30MM (Olympus, model: U-IT101 )
    9. BX2 filter cube (U-MF2) for Olympus BX2 and IX2 models (Chroma Technology, model: U-MF2, catalog number: 91018 )
    10. 510 nm beamsplitter (BS, Part Size: 25.5 x 36 x 1 mm) (Chroma Technology, model: T510lpxrxt )
    11. 50/50 beamsplitter (BS, Part Size: 25.5 x 36 x 1 mm) (Chroma Technology, model: 21000 )
    12. EVGA GeForce GT 520 graphic card with a mini HDMI connector (EVGA, model: 01G-P3-1526-KR )
    13. Light-to-voltage optical sensors (ams, TAOS, model: TSL13T )

Procedure

  1. Primary cultures of neurons expressing channelrhodopsin
    Note: Protocol for the culture of primary neurons was originally described by (Brewer et al., 1993) and was further modified by Hilgenberg and Smith (2007) and Barral and Reyes (2016). This procedure requires aseptic conditions for which the Gibco Cell Culture Basics Handbook can provide a good introduction.
    1. Coating of the coverslips (prepare the day before culturing under sterile conditions)
      1. Clean the coverslips in 3 N HCl or concentrated nitric acid (70% wt/wt) for at least 24 h.
      2. Rinse the coverslips 3 times in distilled H2O, clean them with 70% ethanol and let dry under the tissue culture hood.
      3. Coat cleaned coverslips with 0.1 ml of PLL solution (see Recipes) for 12 h.
      4. Rinse 3 times in distilled H2O and let dry.
    2. Dissociation and culture of primary neurons. Depending on the size of the slicing container, 100 to 150 ml of dissection solution is needed (see Recipes).
      1. Cut 5-6 600 µm coronal slices of cortex from postnatal (P0-P1) mice in ice-cold dissection solution.
      2. In the meantime, prepare the papain solution (see Recipes).
      3. Using forceps, remove the thin membrane covering the brain called the meninges. Dissect out the cortex and cut the cortical layer in small pieces of 0.5-1 mm in length. Transfer the brain pieces in clean dissection solution.
      4. From this step on, the procedure has to be performed under a tissue culture hood. Filter the papain solution in a Petri dish and incubate the brain pieces in the papain solution for 20 min at 37 °C.
      5. In the meantime, dilute the DNase/Mg solution (see Recipes) in 10 ml and prepare 4 conical tubes as follow:
        1: 4 ml of DNase/Mg solution
        2: 2 ml of DNase/Mg solution + 200 µl of trypsin inhibitor solution (see Recipes)
        3: 2 ml of DNase/Mg solution + 60 µl of trypsin inhibitor solution
        4: 2 ml of DNase/Mg solution + 30 µl of trypsin inhibitor solution
      6. Using the transfer pipet, transfer the tissue in tube 1 and let the pieces settle down. Try to transfer the brain tissue with the smallest possible volume of solution. Repeat with tubes 2-4.
      7. Transfer the pieces in about 0.6 ml of equilibrated culture medium (see Recipes) in a low retention tube. Pipet 4-5 times with a P1000 low retention pipet and let the large pieces settle down. Transfer the pellet to a second low retention tube containing about 0.6 ml of culture medium. Pipet 4-5 times with a P200 low retention pipet and pool the two tubes.
      8. Dilute 10 μl of cell suspension in 10 μl of 0.4% trypan blue and estimate the number of viable cells using a hemacytometer. This is useful to control the final density of neurons.
      9. Plate 0.2-3 x 106 cells on each coverslip, resulting in a density of ~50-1,000 cells/mm2 at the time of experiment. Let settle for 1 h (in the incubator) for cells to adhere before flooding the Petri dish with 3 ml of equilibrated culture medium. Maintain cultures at 37 °C and 5% CO2.
      10. Exchange one third of the medium every 2-3 days. Cultures can be maintained in these conditions for up to 3 weeks (see Figure 1 for representative results).


        Figure 1. Culture of primary cortical neurons at 14 days in vitro. Representative examples of a healthy culture where cell bodies are well defined (A) and a culture where the coverslip coating was not achieved properly which resulted in cell aggregates and debris (B).

    3. Expression of channelrhodopsin
      1. The virus AAV2-hSyn-hChR2(H134R)-mCherry) was produced at 3 x 1012 cfu/ml by the University of North Carolina Vector Core Services using plasmid generously provided by Karl Deisseroth (Stanford University). The virus could also be produced in house using standard protocol (Luo et al., 2007).
      2. Infect each Petri dish with 1 µl of virus after 3 days in vitro.
      3. Experiments can be performed between 14-21 DIV, when neuronal characteristics and network connectivity were stable and expression of ChR2 was sufficient to enable reliable photostimulation.

  2. Building and calibrating the photostimulation setup
    The setup for optical stimulation using a video projector was originally described in (Stirman et al., 2012) and further modified in (Barral and Reyes, 2016). The reader will find much details of the procedure to combine a video projector with the microscope in (Stirman et al., 2011; Stirman et al., 2012). The general idea is to focus the image of the projector in the same plane as the sample. Because our microscope was mounted on a movable platform, the photostimulation setup had to be physically connected to the microscope. We describe here the procedure to achieve this but alternative solutions are available and might be more appropriate depending on the setup configuration. Here, we attached a breadboard onto the fluorescent path of the microscope to build the photostimulation light path. We chose the breadboard small enough to limit weight on the microscope but it forced us to use an additional mirror. If possible, try to avoid this mirror by having a longer straight path to project the light coming from the projector.
    1. Building the stimulation setup
      1. Attach the dichroic mirror DM1 to the cube and insert it in the dual port. Place the eyepoint adjuster and the dual port above the fluorescent path and below the camera tube lens of the microscope (Figure 2A).
      2. Fix the aluminum breadboard on the fluorescent tube and define the optical path. Make use of a reflective mirror (Figure 2A, RM) if necessary.
      3. Place the Ø2" lens that will play the role of the tube lens (Figure 2B, PTL) as close as possible to the dichroic mirror and evaluate the position of the primary image plane.
      4. Place the DLP projector and the Ø1" zoom lens (Figure 2B, ZL) to form an image of the projector exactly at this location. It might be useful to mount the projector tube lens on a movable track in order to finely adjust its position when the microscope objective is changed for example.


        Figure 2. Photostimulation setup. A. Picture of the photostimulation setup; B. Schematic of the light path. DLP, Digital Light Processing projector; ZL, zoom lens; PTL, projector tube lens; EPA, eyepoint adjuster; CTL, camera tube lens; DM, dichroic mirror; RM, reflective mirror.

      5. It is likely that the collimated beam will be larger than the dual port entry of the microscope. Install the photodiode next to the entry port of the microscope (Figure 3) to collect some of this light from the projector that will be used as a trigger to synchronize photostimulation and electrophysiological recordings. If the light beam is smaller than the entry port, place a 10/90 beamsplitter in-between the projector tube lens (PTL) and the dichroic mirror (DM1) to collect some light from the projector and redirect it to the photodiode system.


        Figure 3. Photodiodes system. The photodiodes are placed next to the entry port of the microscope and collect a fraction of the beam coming from the projector (dashed line).

      6. For recording of neuronal activity, we used patch-clamp recordings of up to 4 neurons in cell-attached and whole-cell configurations to monitor spikes and post-synaptic potentials, respectively. A useful summary of patch-clamp procedure can be found on Axolbio webpage. Alternatively, the setup is fully compatible with the use of microelectrode array (MEA) recordings if many neurons need to be recorded. Different commercial MEA systems are available or an open-source DIY system called NeuroRighter can be built for a lower cost.
    2. Calibrating the setup
      1. Brightness and pixel size
        1. Project a full-on pattern and measure the total light flux using a light power meter (Figure 4A).
        2. Measure the area of projection using a calibrated camera to deduce the resulting pixel size and the light intensity in mW/mm2. With the maximum current to the blue LED, we achieved 15 mW/mm2.


          Figure 4. Calibration of the photostimulation setup. Images sent to the projector (left) and resulting images from the camera (right) when a full-on (A), ANSI checkerboard (B), or a 4 x 4 white dots (C) pattern were projected.

      2. Contrast ratio
        Here we want to measure the light intensity at the point of stimulation and at other locations to estimate the contrast ratio. This measure is important because it provides an estimate of the light intensity that a non-stimulated neuron receives during stimulation of ChR2 expressing neurons.
        1. Place a 50/50 beamsplitter at the place of DM1 and place a 100% reflecting mirror at the sample plane and use the camera to measure the pixel intensity.
        2. Use a camera to measure the average pixel brightness of a white and of a black test pattern. Estimate the contrast ratio using the full-on full-off method measurements as a ratio of white to black. We measured 815:1. Use the faintest LED current for this procedure to avoid saturation of the camera.
        3. Project an ANSI checkerboard pattern composed of 16 rectangles, eight white and eight black (Figure 4B). Measure the ANSI contrast ratio as the quotient of the averaged white pixels to the black pixels. We measured 21:1, compared to the 43:1 value that was provided by the manufacturer of the projector. It gives a lower bound of the contrast ratio since a large amount of pixels (half of them) are ON in this configuration.
        4. To measure the real contrast ratio during experiment, namely to estimate background illumination of the system, we measured the contrast ratio when a single region of interest was illuminated or when 20 areas were simultaneously illuminated. We found the respective values of 700:1 and 170:1. During an actual experiment with a light intensity of 10 mW/mm2, the background light intensity is therefore between 10 and 50 µW/mm2, which would give rise to photocurrent of about 8-32 pA. This current is not sufficient to evoke spikes but can still elicit postsynaptic-like potentials on the order of 0.1 to 1 mV. However, the largest estimate of background stimulation is only relevant when neurons are activated simultaneously.
      3. Relation between projector and camera pixels
        1. Project a pattern of 4 x 4 white dots evenly spaced on a black background (Figure 4C).
        2. Measure their positions on the camera.
        3. Knowing the position of the dots on the projected pattern and their locations on the image from the camera, use interpolations techniques to map every pixel of the camera to every pixel of the projector.
        4. This procedure can be achieved either by placing a mirror at the sample plane. One can also simply look at the reflection of the projector on the glass coverslip of the sample, which is useful if one wants to run this calibration during an actual experiment.
        5. Draw an arbitrary ROI, build the corresponding pattern, project and image it to assess the quality of the calibration.
    3. Interfacing the projector and the computer
      The projector was controlled by the computer through a USB-based connection. One display mode (‘pattern sequence’ mode) enabled displaying a limited number of single-bit pattern at a rate up to 4 kHz. With the DLP lightcrafter, only 96 patterns can be stored. In this configuration, the predefined patterns were loaded into the remote memory of the projector and displayed using an external analog trigger. More recent projectors can have additional memory but it will never be sufficient to display patterns at high rate for more than few seconds. Additionally, the time to transfer data to the projector can be prohibitive.
      To increase the number of patterns, an alternative method (‘HDMI video’ mode) was used to stream the images through a high speed HDMI connection. Here, images were sent continuously from the computer to the projector via the graphic card using the HDMI port. We used the ‘monochrome 1-bit per pixel’ video mode of the projector. This mode allows bypassing any video processing algorithms. A 24-bits RGB image is then considered as a succession of 24 single-bit planes. The resulting time resolution becomes 24 x 60 Hz = 1,440 Hz for a single-bit image, but without any limitation on the number of displayed patterns.
      Synchronizing accurately the display of each frame with the acquisition of electrophysiological signals was a complicated task because it depends on the precise time at which the computer sends an image to the projector. Since the trigger present on the video projector was already used as an input trigger for the ‘pattern sequence’ mode, we chose to directly measure the light intensity at the output of the projector using a photodiode. Because, the collimated beam was slightly larger than the entry port of the microscope, we could place the photodiode at the edge of the aperture to capture light that did not enter the microscope (see Figure 3). Due to hardware specifications, there is a small 135 µsec delay for every 24-bits plane (i.e., every 16.7 msec) where the image becomes completely dark. We took advantage of this delay, which serves as an accurate trigger for synchronizing photostimulation and electrophysiological recordings offline.
      1. Install the graphic card.
      2. Install the Graphical User Interface (GUI) provided by Texas Instrument and follow the instructions. Connect the projector using the USB and the HDMI connectors.
      3. Define the projector as an external display of the computer.

  3. Independent stimulation of neurons using patterned illumination and recording of neuronal activity
    1. Transfer the neuronal culture coverslip on the microscope chamber and flood with artificial cerebrospinal fluid (see Recipes).
    2. Take a fluorescent image of the neuronal culture (Figure 5).


      Figure 5. Pattern stimulation of the neuronal culture. 36 regions of interest (ROIs) are drawn around stimulated neurons (blue squares). 3 patch-clamp electrodes record spikes and/or subthreshold membrane potential of non-stimulated neurons.

    3. Define regions of interest (ROIs) onto neurons that will be activated (Figure 5).
    4. Using a custom-built interface (Labview or Matlab are suitable for this task), build a temporal sequence of images in order to stimulate each ROI with the appropriate temporal pattern (see Table 1 for the key elements of the interface).

      Table 1. Key elements that the custom-built interface must be able to achieve


    5. Compile a movie from this sequence of images.
      Note: Remember to save the movie without any compression since it would destroy the desired pattern sequence.
    6. Pull recording pipets (aim for a pipet resistance of 5-8 MΩ) and fill patch-clamp electrodes with the internal solution (see Recipes).
      Note: Here recordings were made in current-clamp configuration, therefore a high pipet resistance was acceptable and had the advantage of being easier to form a seal.
    7. Select a neuron. Patch the neuron and record the electrophysiological signal in cell-attached or whole-cell configuration.
      Note: Add QX-314 (Na+ channel blocker) to block spikes if excitatory or inhibitory postsynaptic potentials need to be isolated.
    8. Display the stimulation movie through the projector (see Video 1) and record both the electrophysiological signal and the light intensity from the photodiode. Here, signals were filtered at 5 kHz and digitized at 25 kHz using an 18-bit interface card.

      Video 1. Pattern stimulation of the neuronal culture. 36 regions of interest (ROIs) are drawn around stimulated neurons (blue squares). Each stimulated neuron is activated by a train of light pulses (the actual stimulus is shown in blue on the top left corner). Three neurons are recorded simultaneously in cell-attached mode (yellow raster plots of evoked spikes in each corner, 15 repetitions of the stimulus) and then in whole-cell configuration (yellow trace of membrane potential, average of 7 repetitions).

    9. Using the data from the photodiode, determine the precise time point where the light stimulation actually started. Realign the electrophysiological to this true starting point and analyze data offline.

Data analysis

  1. Control experiment
    To assess the effectiveness of the photostimulation setup in evoking spikes, we stimulated ChR2-positive neurons. We verified that spikes were evoked reliably when a single neuron was stimulated (Figure 6A). Then we stimulated 11 neurons simultaneously and recorded spikes from 2 of them (Figure 6B). Whereas one neuron responded only when stimulated by the light pulses, the other neuron also displayed action potentials in response to the stimulation of neighboring neurons, meaning that it was an integrant component of the recurrent network.


    Figure 6. Pattern stimulation efficacy in evoking spikes in ChR2-expressing neurons. In the following schematics, neurons expressing ChR2 are shown in light red. Recorded neurons are designated by the recording pipets. Light-stimulated neurons have colored contours. A. A train of action potentials (black dots) was applied to two different neurons expressing ChR2 (blue and orange) independently (top and bottom, respectively). Several trials were realized to confirm the faithful stimulation of neurons of interest. Colored dots denote recorded action potential and grey lines the light flashes. No action potentials were observed in the non-stimulated neuron. B. Then, we stimulated 11 neurons in the network. We generated 11 spike trains (top) that were applied to neurons expressing ChR2, including the two previous neurons. Here, we used correlated spike trains for the stimulus (the average correlation between spike trains was C = 0.5). The blue and the orange neurons were recorded simultaneously.

  2. Network stimulation
    The final experiment consists in stimulating the selected neurons by a controlled stimulus (Figure 7A). Here, the firing rate of the input was fixed at 5 Hz and the spike trains were not correlated. These quantities can be varied at will by the experimenter. Neuronal activity in non-stimulated neurons was monitored either in cell-attached (Figure 7B) or in whole-cell (Figure 7C) configuration to isolate the spiking activity and the postsynaptic inputs, respectively. Cell-attached recordings (Figure 7B) showed that some cells fired robustly (neuron 2) while little activity was evoked in others (neurons 1 and 3). Subsequent whole-cell recordings in the same neurons also showed that large voltage transients evoked reliable action potentials across trials. The amplitudes were much larger than the unitary EPSPs and were mostly likely due to synchronous recurrent activity (Barral and Reyes, 2016).


    Figure 7. Independent stimulation of the neuronal network. A. Spike trains which are used for stimulating the 36 selected neurons in Figure 5. Each line is a train of light pulses applied to a given ROI. B. Raster plot of three neurons recorded simultaneously. Each line represents a given repetition of the same stimulus (15 repetitions). C. Corresponding membrane potential of Cell 2. Each repetition (7 repetitions) is drawn as a grey line and the average is displayed in red. The patch pipet contained the Na+ channel blocker QX-314 to block spikes and isolate postsynaptic potentials.

Notes

  1. The protocol to culture primary cortical neurons can work equally on hippocampal neurons.
  2. Add 5 μM Ara-C or 5 μM 5-Fluoro-2’-deoxyuridine on day 3-5 if glial cells show excessive proliferation. However, it is usually not necessary since Neurobasal culture medium with B-27 supplement does not promote glia cell proliferation and it might increase cell death.
  3. We described here a setup using a 460 nm light source to stimulate ChR2-expressing neurons. The setup can easily be modified to use another LED of the projector. Alternatively, the light engine of the projector can be dismounted and an external light source can be used to enlighten the digital micromirror device.

Recipes

  1. Poly-L-Lysine (1x, 0.1 mg/ml PLL, 20 aliquots of 0.5 ml)
    1. Make 100 ml of boric acid/sodium tetraborate solution (0.1 M, pH 8.4)
      Dissolve 0.95 g sodium tetraborate decahydrate in 100 ml ddH2O
      Add boric acid to borax solution until desired pH (pH 8.4) is reached (about 0.61 g of boric acid)
    2. Dissolve 1 mg of PLL in 10 ml boric acid/sodium tetraborate solution
    3. Filter through 0.2 μm, make 0.5 ml aliquots and store at -20 °C for up to 6 months
  2. Dissection solution (1x, CMF-HBSS/HEPES with antibiotics, 1 L)
    Note: Ca2+ and Mg2+ free Hank’s balanced salt solution containing 1 mM pyruvate,15 mM HEPES, 10 mM NaHCO3 and antibiotics.
    1. Mix
      100 ml of 10x CMF-HBSS
      3.9 g HEPES (15 mM final)
      0.84 g NaHCO3 (10 mM final)
      10 ml 100x sodium pyruvate (1 mM final)
      10 ml 100x penicillin/streptomycin (100 U/ml penicillin/100 μg/ml streptomycin final)
      800 ml of distilled H2O
    2. Adjust pH to 7.2 with 1 N NaOH and add H2O to 1 L
    3. Filter sterilize through 0.2 μm sterile filter, store up to 1 month at 4 °C if the pH remains stable
  3. Papain solution (1x, 15 U/ml papain, 100 U/ml DNase, 2 ml)
    1. Add 30 U papain (i.e., 100 µl) to 1.8 ml of dissection solution and warm up to 30-32 °C for 20 min to clear the solution before use
    2. Add 0.2 ml of DNase/L-cystein solution (containing 200 U of DNase and 0.4 mg of L-cysteine)
    3. Adjust pH to 7.4 with about 5 μl of 0.1 N NaOH
    4. Filter through 0.2 μm and use it right away
  4. DNase/L-cysteine solution for papain solution (20x, 100 U/ml DNase final, 1.8 mM L-cystein final, 10 aliquots of 0.2 ml)
    1. Dissolve 2,000 U (1 mg) of DNase and 6.4 mg of L-cysteine in 2 ml of dissection solution
    2. Make 0.2 ml aliquots and store at -20 °C for up to 6 months
  5. DNase/Mg solution for dissociation (50x, 20 U/ml DNase final, 10 aliquots of 0.2 ml)
    1. Dissolve 2,000 U (1 mg) of DNase, 81.3 mg of MgCl2 (2 mM final) and 14.7 mg CaCl2 (1 mM final) in 2 ml of dissection solution
    2. Filter through 0.2 µm, make 0.2 ml aliquots and store at -20 °C for up to 6 months
  6. Trypsin inhibitor solution (100 mg/ml BSA, 40 mg/ml trypsin inhibitor)
    1. In 3 ml of dissection solution (or CMF-HBSS), dissolve 0.3 g BSA (Bovine Albumin) and 0.12 g of trypsin inhibitor
    2. Adjust pH to 7.4 with 1 N NaOH
    3. Filter through 0.2 µm, make 0.3 ml aliquots and store at -20 °C for up to 6 months
  7. Culture medium (NB/B27 medium)
    1. Mix
      485 ml of NBM
      10 ml of B27
      1.25 ml GlutaMAX
      5 ml 100x penicillin/streptomycin (100 U/ml penicillin/100 μg/ml streptomycin final)
    2. Filter through 0.2 μm filter, aliquot in 50 ml and store at 4 °C
  8. Artificial cerebrospinal fluid (1x)
    125 mM NaCl
    10 mM NaHCO3
    25 mM D-glucose
    2.5 mM KCl
    2 mM CaCl2
    1.25 mM NaH2PO4
    1 mM MgCl2
    10 mM HEPES
  9. Intracellular solution (1x)
    130 mM K-gluconate
    10 mM HEPES
    10 mM phosphocreatine
    5 mM KCl
    1 mM MgCl2
    4 mM ATP-Mg
    0.3 mM GTP

Acknowledgments

Jeremie Barral was supported by a Human Frontier Science Program long-term postdoctoral fellowship (LT000132/2012) and by the Bettencourt Schueller Foundation. Alex Reyes was supported by grants from the National Institutes of Health (DC005787-01A1). This protocol was adapted from procedures published in Barral and Reyes (2016).

References

  1. Anselmi, F., Banerjee, A. and Albeanu, D. F. (2015). Patterned photostimulation in the brain. In: Douglass, A. D. (Ed.). New Techniques in Systems Neuroscience. Springer 235-270.
  2. Barral, J. and Reyes, A. (2016). Synaptic scaling rule preserves excitatory-inhibitory balance and salient neuronal network dynamics. Nat Neurosci 19(12): 1690-1696.
  3. Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G. and Deisseroth, K. (2005). Millisecond-timescale, genetically targeted optical control of neural activity. Nat Neurosci 8(9): 1263-1268.
  4. Brewer, G. J., Torricelli, J. R., Evege, E. K. and Price, P. J. (1993). Optimized survival of hippocampal neurons in B27-supplemented neurobasal, a new serum-free medium combination. J Neurosci Res 35(5): 567-576.
  5. Farah, N., Reutsky, I. and Shoham, S. (2007). Patterned optical activation of retinal ganglion cells. Conf Proc IEEE Eng Med Biol Soc 2007: 6368-6370.
  6. Guo, Z. V., Hart, A. C. and Ramanathan, S. (2009). Optical interrogation of neural circuits in Caenorhabditis elegans. Nat Methods 6(12): 891-896.
  7. Hilgenberg, L. G. and Smith, M. A. (2007). Preparation of dissociated mouse cortical neuron cultures. J Vis Exp(10): 562.
  8. Luo, J., Deng, Z. L., Luo, X., Tang, N., Song, W. X., Chen, J., Sharff, K. A., Luu, H. H., Haydon, R. C., Kinzler, K. W., Vogelstein, B. and He, T. C. (2007). A protocol for rapid generation of recombinant adenoviruses using the AdEasy system. Nat Protoc 2(5): 1236-1247.
  9. Nawrot, M. P., Schnepel, P., Aertsen, A. and Boucsein, C. (2009). Precisely timed signal transmission in neocortical networks with reliable intermediate-range projections. Front Neural Circuits 3: 1.
  10. Papagiakoumou, E., Anselmi, F., Begue, A., de Sars, V., Gluckstad, J., Isacoff, E. Y. and Emiliani, V. (2010). Scanless two-photon excitation of channelrhodopsin-2. Nat Methods 7(10): 848-854.
  11. Shoham, S., O’Connor, D. H., Sarkisov, D. V. and Wang, S. S. (2005). Rapid neurotransmitter uncaging in spatially defined patterns. Nat Methods 2(11): 837-843.
  12. Stirman, J. N., Crane, M. M., Husson, S. J., Gottschalk, A. and Lu, H. (2012). A multispectral optical illumination system with precise spatiotemporal control for the manipulation of optogenetic reagents. Nat Protoc 7(2): 207-220.
  13. Stirman, J. N., Crane, M. M., Husson, S. J., Wabnig, S., Schultheis, C., Gottschalk, A. and Lu, H. (2011). Real-time multimodal optical control of neurons and muscles in freely behaving Caenorhabditis elegans. Nat Methods 8(2): 153-158.

简介

我们研究了文化中的皮层神经元网络,并开发了一种创新的光学装置,以空间和时间精确度激发大量神经元。 我们首先描述如何培养表达channelorhodopsin的原代神经元。 然后,我们将根据快速数字光处理(DLP)投影机的工作原理来详细说明光遗传设置。 该设置能够用独立的光脉冲训练数十到数百个神经元,以高时间分辨率诱发动作电位。 在光刺激期间,使用多达4个神经元的膜片钳记录监测网络活动。 该实验非常适合研究复杂的网络动力学或生物过程,如神经元网络中的可塑性或体内平衡,当子群体由其特征(相关性,速率和大小)进行精细控制的不同刺激激活时。
【背景】光致遗传学提供以毫秒精度控制神经元活动的平均值。然而,神经元通常通过同时激活整个群体的光的闪光或通过在整个视野上的时间调制强度的光同时激活(Boyden等人,2005)。然而,存在几种空间调节光并已被用于使谷氨酸不起作用的方法(Nawrot等人,2009)或激活表达神经元的通道视紫质(ChR2)(Guo等人,2009)(用于审查刺激神经元的可用方法具有空间和时间分辨率参见Anselmi等人,2015)。
为了获得刺激的空间控制,第一种可能性是使用激光并将其光束快速移动到不同位置。例如,通过用声光偏转器偏转激光束已经实现了在不同树枝状位置处的谷蛋白解冻(Shoham等人,2005)。只有我们在有限的区域内足够缓慢地调节光强度,这个策略才可能是可行的。或者,可以使用相位或强度的光调制器来实现光的空间图案。基于相位调制的全息技术允许以三维空间精度获得图像,但是可以以仅100Hz的速率显示图案(Papagiakoumou等人,2010)。如果二维图案是足够的,则可以通过将投影仪或阵列的LED放置在样品的共轭平面中来简单地获得强度调制(Farah等人,2007; Guo等人,2009)。这种技术具有易于实现的优点,可以同时瞄准许多感兴趣的区域,并具有最快的时间分辨率。
在这里,我们利用了基于数字微镜器件(DMD)工作的快速视频投影机。 LED光源由微镜阵列分开,可以以毫秒的精度进行控制,以显示任意的任意图案(Barral和Reyes,2016)。投影机的图像通过一对镜头和显微镜物镜聚焦在样品平面上。 DMD技术提供了前所未有的时间精度,使得能够以1.44 kHz显示图案,甚至更快的DMD可用。在我们的设置中,所得到的像素尺寸(2.2×1.1μm)足够小以刺激单个神经元。
为了激活单个神经元,我们选择了一个感兴趣的区域,约30 x 30μm,以感兴趣的神经元的神经元为中心,发出5毫秒的光脉冲。通过设计投影到样本上的图案,我们可以独立地同时定位大量的神经元(10到100个神经元)。刺激的神经元都是兴奋性和抑制性(在Synapsin启动子下的ChR2表达),并被Poisson穗列车激活。刺激刺激的速率和相关性由实验者控制(参见Barral和Reyes,2016)。通过从表达ChR2的神经元进行记录,我们验证了刺激的神经元忠实地响应于光脉冲。然后我们同时记录细胞附着和全细胞构型中多达4个神经元的膜电位,分别分离刺激活性和突触后输入。

关键字:神经元的原代培养, 光遗传学, 模式化光学刺激

材料和试剂

  1. 对于神经元的文化
    1. 圆形玻片,直径25毫米,德国玻璃(电子显微镜科学,目录号:72196-25)
    2. 硅胶低保留微量离心管(1.5ml)(Fisher Scientific,目录号:02-681-331)
    3. 低保留移液管吸头200μl(Fisher Scientific,目录号:02-717-165)
    4. 低保留移液管吸头1,000μl(Fisher Scientific,目录号:02-717-166)
    5. 一次性培养皿(35 x 10毫米)(康宁,猎鹰,目录号:351008)
    6. Stericup-GP无菌真空过滤系统,0.22μm,聚醚砜(EMD Millipore,目录号:SCGPU05RE)
    7. 注射器过滤器; MCE膜;孔径:0.22μm(EMD Millipore,目录号:SLGS033SS)
    8. 无菌转移吸管(Fisher Scientific,目录号:13-711-20)
    9. 锥形无菌聚丙烯离心管(Thermo Fisher Scientific,Thermo Scientific TM,目录号:339650)
    10. 产后(P0-P1)小鼠
    11. 盐酸(HCl)(Sigma-Aldrich,目录号:320331)
    12. 硝酸(Sigma-Aldrich,目录号:258121)
    13. 70%乙醇
    14. 聚-L-赖氨酸氢溴酸盐 - 摩尔重量70,000-150,000(Sigma-Aldrich,目录号:P1274)
    15. 四水合硼酸钠≥99.5%(Sigma-Aldrich,目录号:B9876-500G)
    16. 硼酸(测试细胞培养)(Sigma-Aldrich,目录号:B9645-500G)
    17. HBSS(10x),无钙,无镁,无酚红(Thermo Fisher Scientific,Gibco TM,目录号:14185052)
    18. 青霉素 - 链霉素(10,000U/ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
    19. HEPES(Thermo Fisher Scientific,Gibco TM ,目录号:15630080)
    20. 琼脂(Sigma-Aldrich,目录号:A1296-100G)
    21. 碳酸氢钠(NaHCO 3)(Fisher Scientific,目录号:S233)
    22. 丙酮酸钠溶液(Sigma-Aldrich,目录号:S8636-100ML)
    23. 氢氧化钠(NaOH)(Fisher Scientific,目录号:S318)
    24. L-半胱氨酸盐酸盐一水合物(Sigma-Aldrich,目录号:C7880-500MG)
    25. DNase I等级II,来自牛胰腺-100mg(Roche Diagnostics,目录号:10104159001)
    26. 番木瓜木瓜蛋白酶10毫升100mg(Roche Diagnostics,目录号:10108014001)
    27. 氯化钙(CaCl 2)(Fisher Scientific,目录号:C79)
    28. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
    29. 来自鸡蛋白的胰蛋白酶抑制剂(Sigma-Aldrich,目录号:T9253-500MG)
    30. 来自牛血清的白蛋白(粉末,适用于细胞培养,= 96%)(Sigma-Aldrich,目录号:A9418-5G)
    31. 台盼蓝溶液(0.4%,液体,无菌过滤,适合细胞培养)(Sigma-Aldrich,目录号:T8154-20ML)
    32. Neurobasal培养基(1x)(Thermo Fisher Scientific,Gibco TM,目录号:21103049)
    33. 含有维生素A(50x),液体(Thermo Fisher Scientific,Gibco TM,目录号:17504044)的B-27无血清补充剂。
    34. GlutaMAX TM 补充(Thermo Fisher Scientific,Gibco TM,目录号:35050061)
    35. AAV病毒用于ChR2表达(北卡罗来纳大学矢量核心服务部AAV2-hSyn-hChR2(H134R)-mCherry)
    36. 氯化钠(NaCl)(Fisher Scientific,目录号:BP358)
    37. D-葡萄糖(Fisher Scientific,目录号:BP350)
    38. 氯化钾(KCl)(Fisher Scientific,目录号:P333)
    39. 磷酸二氢钠(NaH 2 PO 4)(Fisher Scientific,目录号:S369)
    40. K - 葡萄糖酸盐(Sigma-Aldrich,目录号:P1847)
    41. 磷酸肌酸(Sigma-Aldrich,目录号:P1937)
    42. ATP-Mg(Sigma-Aldrich,目录号:A9187)
    43. GTP(Sigma-Aldrich,目录号:G8877)
    44. 聚-L-赖氨酸(PLL溶液)(参见食谱)
    45. 解剖解决方案(见配方)
    46. 木瓜蛋白酶溶液(参见食谱)
    47. DNase/L-半胱氨酸溶液(参见食谱)
    48. DNase/Mg溶液(参见食谱)
    49. 胰蛋白酶抑制剂溶液(参见食谱)
    50. 培养基(NB/B27培养基)(参见食谱)

  2. 电生理记录
    1. 硼硅玻璃毛细管(1.5 OD)(世界精密仪器,目录号:1B150F-4)
    2. 人造脑脊液(aCSF,见食谱)
    3. 细胞内溶液(见食谱)

设备

  1. 对于神经元的文化
    1. 组织文化罩
    2. P1000移液器
    3. P200移液器
    4. 细胞培养培养箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:Forma TM Series II 3110,目录号:3110)
    5. Vibratome切片机(Leica Biosystems,型号:Leica VT1200 S)
    6. Dumont#5镊子(精细科学工具,目录号:11251-30)
    7. Dumont#7镊子(Fine Science Tools,目录号:11271-30)
    8. (精细科学工具,目录号:14084-08)

  2. 对于电生理设置
    1. 具有荧光的立式水浸式显微镜(Olympus,型号:BX51)
    2. mCherry荧光过滤器(TRITC,Chroma Technology,型号:41002c)
    3. 用于荧光成像的CCD相机(Hamamatsu Photonics,型号:C8484)
    4. 用于红外成像的CCD摄像机(Olympus,型号:OLY-150 IR)
    5. 微操纵器(Luigs& Neumann,型号:SM6)
    6. 贴片放大器(Dagan,型号:BVC-700A)
    7. 18位接口卡(National Instruments,型号:PCI-6289)
    8. 火焰/棕色微量吸管拔出器(Sutter Instrument,型号:P-97)
    9. 血细胞计数仪(Hausser,目录号:3110)

  3. 对于光发现设置
    1. DLP投影机(德州仪器,型号:DLP ® LightCrafter TM 评估模块)
    2. 铝面包板,150 x 300 x 12.7 mm,双密度,M6螺纹(Thorlabs,型号:MB1530/M)
    3. Ø1"消色差双色,SM1螺纹安装,f = 35 mm,ARC:400-700 nm(Thorlabs,型号:AC254-035-A-ML)
    4. Ø2"消色差双色,SM2螺纹安装,f = 200 mm,ARC:400-700 nm(Thorlabs,型号:AC508-200-A-ML)
    5. Ø2"(Ø50.8mm)保护银镜,0.47"(12.0 mm)厚(Thorlabs,型号:PF20-03-P01)
    6. Ø2"光学运动支架(Thorlabs,型号:KM200)
    7. U型DP;双口中间管(Olympus,型号:U-IT140)
    8. U型EPA2; Eyepoint调节器,BX2,提升目标30MM(Olympus,型号:U-IT101)
    9. 用于Olympus BX2和IX2型号的BX2滤镜(U-MF2)(Chroma Technology,型号:U-MF2,目录号:91018)
    10. 510 nm分光镜(BS,部件尺寸:25.5 x 36 x 1 mm)(Chroma Technology,型号:T510lpxrxt)
    11. 50/50分光镜(BS,部件尺寸:25.5 x 36 x 1 mm)(Chroma Technology,型号:21000)
    12. EVGA GeForce GT 520图形卡,配有迷你HDMI连接器(EVGA,型号:01G-P3-1526-KR)
    13. 光电压光学传感器(ams,TAOS,型号:TSL13T)

程序

  1. 表达通道视紫红质的神经元的原代培养物
    注意:原始神经元培养方案最初由(Brewer等人,1993)描述,并由Hilgenberg和Smith(2007)和Barral和Reyes(2016)进一步修改。此过程需要 Gibco细胞培养基础手册可以提供一个很好的介绍。
    1. 盖玻片的涂层(在无菌条件下培养前的一天准备)
      1. 用3N HCl或浓硝酸(70%wt/wt)清洁盖玻片至少24小时。
      2. 在蒸馏的H 2 O 2中冲洗盖玻片3次,用70%乙醇清洗,并在组织培养罩下干燥。
      3. 用0.1毫升PLL溶液清洁盖玻片(参见食谱)12小时。
      4. 在蒸馏的H 2 O 3中冲洗3次并干燥。
    2. 原发神经元的分离和培养。根据切片容器的大小,需要100至150毫升的解剖解决方案(参见食谱)。
      1. 在冰冷的解剖解决方案中从产后(P0-P1)小鼠切下5-6600μm冠状切片的皮层。
      2. 同时,准备木瓜蛋白酶溶液(参见食谱)。
      3. 使用镊子,去除覆盖脑的薄膜,称为脑膜。解剖出皮质并切割长度为0.5-1毫米的小块皮质层。将脑片转移到清洁解剖解决方案中。
      4. 从该步骤开始,该程序必须在组织培养罩下进行。过滤培养皿中的木瓜蛋白酶溶液,并将木瓜蛋白酶溶液中的脑片在37℃下孵育20分钟。
      5. 同时,稀释DNase/Mg溶液(参见食谱)10 ml,制备4个锥形管,如下所示:
        1:4ml DNase/Mg溶液
        2:2ml DNase/Mg溶液+200μl胰蛋白酶抑制剂溶液(参见食谱)
        3:2ml DNase/Mg溶液+60μl胰蛋白酶抑制剂溶液
        4:2ml DNase/Mg溶液+30μl胰蛋白酶抑制剂溶液
      6. 使用转移吸管,转移管1中的组织,并使块沉降。尝试用尽可能小的溶液体积转移脑组织。用管2-4重复。
      7. 将约0.6ml平衡的培养基(见食谱)转移到低保留管中。用P1000低速移液管吸取4-5次,让大块沉降。将颗粒转移到含有约0.6ml培养基的第二个低保留管中。用P200低速移液管吸管4-5次,并将两根管子放在一起。
      8. 稀释10μl细胞悬浮液10μl的0.4%台盼蓝,并使用血细胞计数器估计活细胞数。这对于控制神经元的最终密度是有用的
      9. 在每个盖玻片上放置0.2-3×10 6个细胞,在实验时产生约50-1,000个细胞/mm 2的密度。放置1 h(在培养箱中)使细胞粘附,然后用3 ml平衡的培养基淹没培养皿。维持37℃和5%CO 2的培养物。
      10. 每2-3天交换三分之一的培养基。文化可以在这些条件下维持长达3周(见图1的代表性结果)。


        图1.体外14天初次皮层神经元的培养 健康培养物的代表性实例,其中细胞体定义明确(A)和培养,其中盖玻片未能正确实现,导致细胞聚集和碎屑(B)
    3. 通道视紫红质的表达
      1. 使用Karl Deisseroth(斯坦福大学)慷慨提供的质粒,由北卡罗来纳大学矢量核心服务以3×10 12 cfu/ml生成病毒AAV2-hSyn-hChR2(H134R)-mCherry) 。病毒也可以使用标准协议在家里生产(Luo等人,2007)。
      2. 在体外3天后用1μl病毒感染每个培养皿。
      3. 实验可以在14-21 DIV之间进行,当神经元特征和网络连通性稳定时,ChR2的表达足以实现可靠的光刺激。

  2. 建立和校准光刺激设置
    最初在(Stirman等人,2012)中描述了使用视频投影仪进行光学刺激的设置,并在(Barral和Reyes,2016)中进一步修改。读者将会在(Stirman等人,2011; Stirman等人,2012)中将视频投影仪与显微镜相结合的程序的细节。一般的想法是将投影机的图像与样品相同。因为我们的显微镜被安装在可移动的平台上,所以光刺激装置必须与显微镜物理连接。我们在这里描述实现这一点的过程,但替代解决方案可用,并且可能更适合取决于设置配置。在这里,我们将一个面包板连接到显微镜的荧光路径上,以构建光刺激光路。我们选择了足够小的面包板来限制显微镜的重量,但是它迫使我们使用额外的镜子。如果可能的话,尽量避免使用更长的直线来投影来自投影机的光线
    1. 建立刺激设置
      1. 将分色镜DM1连接到立方体并将其插入双端口。将眼点调节器和双端口放在荧光通路上方,并在显微镜的摄像管镜头下面(图2A)。
      2. 将铝面板板固定在荧光灯管上并定义光路。如有必要,请使用反光镜(图2A,RM)。
      3. 将Ø2"镜头放在尽可能靠近分色镜的管镜上(图2B,PTL)的角色,并评估主像平面的位置。
      4. 将DLP投影机和Ø1"变焦镜头(图2B,ZL)放在正好位于该位置的投影机镜头的形状上,将投影机管道镜头安装在活动轨道上可能很有用,以便在例如显微镜目标改变了。


        图2.光刺激设置 A.光刺激设置的图片光路示意图。 DLP,数码光处理投影仪; ZL,变焦镜头; PTL,投影管镜; EPA,眼点调节器; CTL,摄像管镜头; DM,分色镜; RM,反光镜。

      5. 准直光束很可能比显微镜的双端口入口大。将光电二极管安装在显微镜进入端口旁边(图3),以收集投影机中的一些这样的光,这些光将用作触发器来同步光刺激和电生理记录。如果光束小于进入口,则在投影仪管透镜(PTL)和分色镜(DM1)之间放置10/90分束器,以收集投影机的一些光线并将其重定向到光电二极管系统。 br />

        图3.光电二极管系统光电二极管放置在显微镜入口旁边,并收集来自投影机的一小部分光束(虚线)。

      6. 为了记录神经元活动,我们使用细胞附着和全细胞配置中多达4个神经元的膜片钳记录来分别监测尖峰和突触后电位。可以在 Axolbio 网页。或者,如果需要记录许多神经元,则该设置与使用微电极阵列(MEA)记录完全兼容。可以使用不同的商业MEA系统或称为 NeuroRighter 可以降低成本。
    2. 校准设置
      1. 亮度和像素大小
        1. 投射一个完整的图案,并使用光功率计测量总光通量(图4A)
        2. 使用校准的相机测量投影面积以推导出所得到的像素大小和光强度(mW/mm <2)。通过蓝色LED的最大电流,我们实现了15 mW/mm 2

          图4.光刺激设置的校准。当全功能(A),ANSI棋盘(B)或一张(A))的图像发送到投影机(左)和相机的图像(右)投影4 x 4个白点(C)图案。

      2. 对比度
        在这里,我们要测量刺激点处的光强度,并在其他位置测量对比度。这个措施很重要,因为它提供了在刺激ChR2表达神经元期间非刺激神经元接收的光强度的估计。
        1. 在DM1的地方放置一个50/50的分光镜,并在样品平面上放置100%的反射镜,并使用相机测量像素强度。
        2. 使用相机测量白色和黑色测试图案的平均像素亮度。使用全开全方法测量来估计对比度,作为白色与黑色的比例。我们测量了815:1。使用最弱的LED电流进行此过程,以避免摄像机饱和。
        3. 投射由16个矩形组成的ANSI棋盘图案,8个白色和8个黑色(图4B)。测量ANSI对比度,作为平均白色像素与黑色像素的商。我们测量了21:1,而投影机制造商提供的43:1的值。它给出了对比度的下限,因为在此配置中,大量像素(其中一半)为ON。
        4. 为了测量实验中的实际对比度,即估计系统的背景照明,我们测量了当感兴趣的单个区域被照亮时或当同时照亮20个区域时的对比度。我们发现各自的值为700:1和170:1。在光强度为10mW/mm 2的实际实验中,背景光强因此在10和50μW/mm 2之间,这将导致光电流约8-32 pA。这个电流不足以引起尖峰,但是仍然可以引发0.1到1mV的突触后样电位。然而,背景刺激的最大估计仅在神经元被同时激活时才相关
      3. 投影机和摄像机像素之间的关系
        1. 在黑色背景上投射4 x 4个白点的图案(图4C)。
        2. 测量相机上的位置。
        3. 了解投影图案上的点的位置及其在相机图像上的位置,使用插值技术将相机的每个像素映射到投影机的每个像素。
        4. 该过程可以通过将样品平面上放置反射镜来实现。还可以简单地看看投影机在样品玻璃盖玻片上的反射,如果在实际实验中想要运行该校准,这是有用的。
        5. 绘制任意投资回报率,构建相应的模式,对项目进行投影并进行形象化,以评估校准的质量。
    3. 投影仪和计算机的接口
      投影机由计算机通过基于USB的连接来控制。一种显示模式("模式序列"模式)使得能够以高达4 kHz的速率显示有限数量的单位模式。使用DLP光标,只能存储96种图案。在此配置中,将预定义的模式加载到投影机的远程存储器中,并使用外部模拟触发器进行显示。更多的投影机可以有额外的内存,但是在几秒钟内以高速率显示图案将是不够的。此外,将数据传输到投影机的时间可能会令人望而却步。
      为了增加模式数量,采用另一种方法("HDMI视频"模式)通过高速HDMI连接来流式传输图像。这里,图像通过使用HDMI端口的图形卡从计算机连续发送到投影机。我们使用投影机的"单色1位每像素"视频模式。此模式允许绕过任何视频处理算法。然后将24位RGB图像视为一系列24位单位平面。所得到的时间分辨率对于单位图像而言变为24 x 60 Hz = 1,440 Hz,但对显示的图案数量没有任何限制。
      准确地同步每个帧的显示与获取电生理信号是一个复杂的任务,因为它取决于计算机将图像发送到投影机的精确时间。由于视频投影机上存在的触发器已经被用作"模式序列"模式的输入触发器,所以我们选择使用光电二极管直接测量投影机输出端的光强度。因为准直光束比显微镜的入口端略大,所以我们可以将光电二极管放置在光圈的边缘,以捕获未进入显微镜的光(见图3)。由于硬件规格,对于图像变得完全黑暗的每24位平面(即,每16.7毫秒),存在小的135微秒延迟。我们利用了这个延迟,它作为离线同步光刺激和电生理记录的准确触发。
      1. 安装图形卡。
      2. 安装德州仪器提供的图形用户界面(GUI),并按照说明进行操作。使用USB和HDMI连接器连接投影机。
      3. 将投影机定义为计算机的外部显示器。

  3. 使用图案照明和记录神经元活动来独立刺激神经元
    1. 将神经元培养盖玻片转移到显微镜室并用人造脑脊液灌洗(参见食谱)。
    2. 拍摄神经元培养的荧光图像(图5)

      图5.神经元培养的模式刺激 36个感兴趣区域(ROI)围绕刺激的神经元(蓝色方块)绘制。 3个膜片钳电极记录非刺激神经元的尖峰和/或亚阈值膜电位
    3. 将感兴趣区域(ROI)定义为将被激活的神经元(图5)
    4. 使用自定义界面(Labview或Matlab适用于此任务),构建一个时间序列的图像,以便以适当的时间模式来激发每个ROI(参见表1关于接口的关键元素)。 >
      表1.定制界面必须能够实现的关键要素


    5. 从这个图像序列编译一部电影。
      注意:请记住保存电影没有任何压缩,因为它会破坏所需的图案序列。
    6. 拉出记录移液器(针对5-8MΩ的吸管电阻),并用内部溶液填充膜片钳电极(参见食谱)。
      注意:这里采用电流钳配置进行记录,因此高吸液管阻力是可以接受的,并且具有更容易形成密封的优点。
    7. 选择一个神经元。 补充神经元并记录细胞附着或全细胞配置中的电生理信号 注意:如果兴奋性或抑制性突触后电位需要隔离,请添加QX-314(Na + 通道阻滞剂)以阻止尖峰。
    8. 通过投影机显示刺激电影(见视频1),并记录光电二极管的电生理信号和光强度。 这里,信号在5kHz被滤波,并使用18位接口卡以25kHz数字化。

      Video 1. Pattern stimulation of the neuronal culture. 36 regions of interest (ROIs) are drawn around stimulated neurons (blue squares). Each stimulated neuron is activated by a train of light pulses (the actual stimulus is shown in blue on the top left corner). Three neurons are recorded simultaneously in cell-attached mode (yellow raster plots of evoked spikes in each corner, 15 repetitions of the stimulus) and then in whole-cell configuration (yellow trace of membrane potential, average of 7 repetitions).

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


    9. 使用来自光电二极管的数据,确定光刺激实际开始的精确时间点。将电生理重新定位到这个真正的起点,并离线分析数据。

数据分析

  1. 控制实验
    为了评估光刺激设置在引发峰值中的有效性,我们刺激了ChR2阳性神经元。我们证实当单个神经元受到刺激时,峰值可以被可靠地诱发(图6A)。然后我们同时刺激了11个神经元,并记录了2个神经元(图6B)。而一个神经元只有在受到光脉冲刺激时才响应,另一个神经元也响应于相邻神经元的刺激而显示动作电位,这意味着它是复发网络的一个整合部分。


    图6.在表达ChR2的神经元中引发尖峰的模式刺激功效在下面的示意图中,表达ChR2的神经元以浅红色显示。记录的移液管指定记录的神经元。光刺激的神经元具有彩色轮廓。 A.一系列动作电位(黑点)分别应用于分别表示ChR2(蓝色和橙色)的两种不同的神经元(分别为顶部和底部)。实现了几项试验,以确认对目标神经元的忠实刺激。彩色点表示记录的动作电位和指示灯闪烁的灰色线。在未刺激的神经元中没有观察到动作电位。然后,我们刺激了网络中的11个神经元。我们生成了11个尖峰火车(上),应用于表达ChR2的神经元,包括前两个神经元。在这里,我们使用相关的穗列刺激(穗火车的平均相关性为C = 0.5)。同时记录蓝色和橙色神经元。

  2. 网络刺激
    最终实验包括通过受控刺激刺激所选择的神经元(图7A)。这里,输入的燃烧速率固定在5Hz,并且尖峰火车不相关。这些数量可以由实验者随意改变。在细胞附着(图7B)或全细胞(图7C)构型中监测非刺激神经元中的神经元活性,以分别分离刺激活性和突触后输入。细胞附着的记录(图7B)显示,一些细胞强壮地发射(神经元2),而在其他细胞中没有诱发活动(神经元1和3)。随后在同一神经元中的全细胞记录也表明,大电压瞬变引起了跨试验的可靠动作电位。幅度比单一EPSP大得多,主要是由于同步的复发活动(Barral和Reyes,2016)。


    图7.神经网络的独立刺激。 A.用于刺激图5中36个选定神经元的尖峰火车。每条线是施加到给定ROI的一串光脉冲。 B.同时记录的三个神经元的光栅图。每行表示相同刺激的给定重复(15次重复)。 C.细胞2的相应膜电位。每个重复(7个重复)被绘制为灰色线,平均值显示为红色。补片移液管包含Na + 通道阻滞剂QX-314以阻断尖峰并分离突触后电位。

笔记

  1. 培养原代皮层神经元的方案可以在海马神经元上同样工作
  2. 如果神经胶质细胞显示过度增殖,则在第3-5天加入5μMAra-C或5μM5-氟-2'-脱氧尿苷。然而,由于具有B-27补充剂的神经巴斯培养基不会促进胶质细胞增殖并且可能增加细胞死亡,通常不是必需的。
  3. 我们在这里描述了使用460nm光源刺激表达ChR2的神经元的设置。该设置可以轻松修改,以使用投影机的其他LED。或者,可以拆下投影机的光引擎,并且可以使用外部光源来启发数字微镜设备。

食谱

  1. 聚-L-赖氨酸(1x,0.1mg/ml PLL,20份0.5ml等分试样)
    1. 制备100ml硼酸/四硼酸钠溶液(0.1M,pH8.4) 将0.95g十水合四硼酸钠溶于100ml ddH 2 O中, 将硼酸加入硼砂溶液中直到达到所需pH(pH8.4)(约0.61g硼酸)
    2. 将1毫克PLL溶于10毫升硼酸/四硼酸钠溶液中
    3. 过滤0.2μm,制备0.5ml等分试样,并在-20℃下储存长达6个月
  2. 解剖解决方案(1x,CMF-HBSS/HEPES与抗生素,1 L)
    注意:含有1mM丙酮酸盐,15mM HEPES,10mM NaHCO 3的游离Hank's平衡盐溶液的Ca 2 + 和Mg 2 + >和抗生素。
    1. 混合
      100毫升10倍CMF-HBSS
      3.9 g HEPES(15 mM final)
      0.84g NaHCO 3(10mM最终)
      10 ml 100x丙酮酸钠(最终1 mM)
      10 ml 100x青霉素/链霉素(100 U/ml青霉素/100μg/ml链霉素最终)
      800毫升蒸馏水H 2 O
    2. 用1N NaOH将pH调节至7.2,并加入H 2 O至1L
    3. 通过0.2μm无菌过滤器过滤消毒,如果pH保持稳定,可在4°C下储存1个月
  3. 木瓜蛋白酶溶液(1x,15U/ml木瓜蛋白酶,100U/ml DNase,2ml)
    1. 将30U木瓜蛋白酶(即,100μl)加入到1.8ml的解剖溶液中并加热至30-32℃20分钟,以在使用前清除溶液
    2. 加入0.2ml DNase/L-半胱氨酸溶液(含有200U的DNase和0.4mg的L-半胱氨酸)
    3. 用约5μl0.1N NaOH调节pH至7.4
    4. 过滤0.2μm,立即使用
  4. 木瓜蛋白酶溶液的DNA酶/半胱氨酸溶液(20x,100U/ml DNA酶终体,1.8mM L-半胱氨酸终体积,10份等分试样为0.2ml)
    1. 将2 000 U(1 mg)DNase和6.4 mg L-半胱氨酸溶解于2 ml解剖溶液中
    2. 制备0.2ml等分试样,并在-20℃下储存长达6个月
  5. 用于解离的DNA酶/Mg溶液(50x,20U/ml DNase最终,10等份的0.2ml)
    1. 将2U溶解于2ml解剖溶液中的2000U(1mg)DNase,81.3mg MgCl 2(最终2mM)和14.7mg CaCl 2(最终1mM)溶解
    2. 过滤0.2μm,制成0.2 ml等分试样,-20℃保存6个月
  6. 胰蛋白酶抑制剂溶液(100mg/ml BSA,40mg/ml胰蛋白酶抑制剂)
    1. 在3ml解剖溶液(或CMF-HBSS)中,溶解0.3g BSA(牛白蛋白)和0.12g胰蛋白酶抑制剂
    2. 用1N NaOH调节pH至7.4
    3. 过滤0.2μm,制成0.3ml等分试样,并在-20℃下储存长达6个月
  7. 培养基(NB/B27培养基)
    1. 混合
      485毫升NBM
      10 ml B27
      1.25 ml GlutaMAX
      5 ml 100x青霉素/链霉素(100 U/ml青霉素/100μg/ml链霉素最终)
    2. 通过0.2μm过滤器过滤,分装在50ml中,并在4°C下储存
  8. 人造脑脊液(1x)
    125 mM NaCl
    10mM NaHCO 3
    25mM D-葡萄糖
    2.5 mM KCl
    2mM CaCl 2
    1.25mM NaH 2 PO 4
    1mM MgCl 2
    10 mM HEPES
  9. 细胞内溶液(1x)
    130 mM K-gluconate
    10 mM HEPES
    10 mM磷酸肌酸
    5 mM KCl
    1mM MgCl 2
    4 mM ATP-Mg
    0.3 mM GTP

致谢

Jeremie Barral得到人类边疆科学计划长期博士后研究金(LT000132/2012)和Bettencourt Schueller基金会的支持。 Alex Reyes由美国国家卫生研究院(DC005787-01A1)提供资助。该协议是根据Barral和Reyes(2016)发布的程序进行的。

参考

  1. Anselmi,F.,Banerjee,A.and Albeanu,DF(2015)。< a class ="ke-insertfile"href ="https://link.springer.com/chapter/10.1007/978-3-319 -12913-6_9"target ="_ blank">大脑中图案化的光刺激。在In:Douglass,AD(Ed。)。系统神经科学中的新技术。 Springer 235-270。
  2. Barral,J.和Reyes,A.(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/27749827"target ="_ blank" >突触定标规则保留兴奋性抑制平衡和显着神经元网络动态。 Nat Neurosci 19(12):1690-1696。
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引用:Barral, J. and Reyes, A. D. (2017). Optogenetic Stimulation and Recording of Primary Cultured Neurons with Spatiotemporal Control. Bio-protocol 7(12): e2335. DOI: 10.21769/BioProtoc.2335.
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