对原代培养神经元进行时空控制的光遗传学刺激和记录

Jérémie  Barral; Alex D Reyes

doi:10.21769/BioProtoc.2335

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Peer-reviewed

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

JB Jérémie Barral email

AR Alex D Reyes

发布: 2017年06月20日第7卷第12期 DOI: 10.21769/BioProtoc.2335 浏览次数: 12189

评审: Pengpeng LiZinan ZhouAnonymous reviewer(s)

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Cover of Nature Neuroscience, featuring study using the protocol.

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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

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 Scientific^TM, 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, Gibco^TM, catalog number: 14185052 )
18. Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
19. HEPES (Thermo Fisher Scientific, Gibco^TM, 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, Gibco^TM, catalog number: 21103049 )
33. B-27 serum-free supplement containing vitamin A (50x), liquid (Thermo Fisher Scientific, Gibco^TM, catalog number: 17504044 )
34. GlutaMAX^TM supplement (Thermo Fisher Scientific, Gibco^TM, 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)
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

For the neuronal culture
1. Tissue culture hood
2. P1000 pipet
3. P200 pipet
4. Cell culture incubator (Thermo Fisher Scientific, Thermo Scientific^TM, model: Forma^TM 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 )
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 )
For the optogenetic setup
1. DLP projector (Texas Instruments, model: DLP^® LightCrafter^TM 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

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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.