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Optogenetic Mapping of Synaptic Connections in Mouse Brain Slices to Define the Functional Connectome of Identified Neuronal Populations
对小鼠脑切片中突触连接进行光遗传学图谱绘制来确定已鉴定的神经元群体的功能连接体   

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Abstract

Functional connectivity in a neural circuit is determined by the strength, incidence, and neurotransmitter nature of its connections (Chuhma, 2015). Using optogenetics the functional synaptic connections between an identified population of neurons and defined postsynaptic target neurons may be measured systematically in order to determine the functional connectome of that identified population. Here we describe the experimental protocol used to investigate the excitatory functional connectome of ventral midbrain dopamine neurons, mediated by glutamate cotransmission (Mingote et al., 2015). Dopamine neurons are made light sensitive by injecting an adeno-associated virus (AAV) encoding channelrhodopsin (ChR2) into the ventral midbrain of DATIREScre mice. The efficacy and specificity of ChR2 expression in dopamine neurons is verified by immunofluorescence for the dopamine-synthetic enzyme tyrosine hydroxylase. Then, slice patch-clamp recordings are made from neurons in regions recipient to dopamine neuron projections and the incidence and strength of excitatory connections determined. The summary of the incidence and strength of connections in all regions recipient to dopamine neuron projections constitute the functional connectome.

Keywords: Channelrhodopsin(通道视紫红质), Dopamine(多巴胺), Cotransmission(共同传递), Patch-clamp(膜片钳), Immunofluorescence(免疫荧光), Adeno associated virus(腺相关病毒)

Background

To establish the function of specific neural circuits it is necessary to determine the anatomical connectome, the mapping of anatomical connections, and its functional connectome, the mapping of the strength, incidence and neurotransmitter nature of connections. The use of viral transsynaptic tracing techniques that are monosynaptically restricted, allows for the description of complex anatomical connections of neural circuits, including the dopamine system (Callaway and Luo, 2015; Faget et al., 2016). The functional connectivity of these circuits has been harder to determine due to the intermingling of axons that make selective electrical stimulation impossible. With the advent of optogenetics it became possible to stimulate genetically defined populations of cells selectively. This allowed for the identification of new connections made by striatal medium spiny neurons (Chuhma et al., 2011), ventral midbrain glutamate neurons (Hnasko et al., 2012; Root et al., 2014) and dopamine/glutamate neurons (Mingote et al., 2015). Moreover, optogenetics used in a systematic and comprehensive manner to map the incidence and strength of connections of specific neuronal populations to defined postsynaptic target regions, determines the functional connectome of defined neuronal populations (Chuhma et al., 2011; Mingote et al., 2015). In this protocol, we describe how to determine the functional connectome of any genetically defined neuronal population. As an example, we focus on the excitatory functional connectome of dopamine neurons, mediated by glutamate cotransmission (Mingote et al., 2015).

Part I: Inducing channelrhodopsin expression in dopamine neurons by viral transfection

Materials and Reagents

  1. Glass PCR micropipettes with 1 μl marks (Drummond Scientific, catalog number: 5-000-1001-X10 )
  2. Kimwipe
  3. Syringe
  4. Q-tip
  5. Surgical blades No.11 (Thomas Scientific, catalog number: 3883B59 )
  6. Needle
  7. Mice (THE JACKSON LABORATORY, Strain 006660: DATIREScre )
    Note: This knock-in mouse expresses cre recombinase under the transcriptional control of the endogenous dopamine transporter (DAT) promoter. To minimize the interference with the DAT promoter function, cre recombinase expression is driven from the 3’ untranslated region via an internal ribosomal entry sequence (IRES).
  8. AAV5-EF1α-DIO-hChR2(H134R)-EYFP (titer: 8x10e-12 virions/ml) (Addgene, catalog number: 20298 ) is used to drive cre-dependent expression of ChR2-EYFP. The adeno-associated virus (AAV) can be obtained under a MTA from Dr. Karl Deisseroth from the vector core at the University of North Carolina; this AAV is serotype 5 and replication-incompetent.
  9. Paraffin
  10. 10% bleach solution
  11. Carprofen (Rimadyl, Zoetis)
  12. Ketamine HCl (KetaVed, Vedco)
  13. Xylazine (Akorn, AnaSed®)
  14. Puralube VET ointment, sterile ocular lubricant (Dechra)
  15. Lidocaine HCl (40 mg/ml) for local anesthesia (Boehringer Ingelheim)
  16. Vetbond, n-butyl cyanoacrylate adhesive (3M)
  17. 70% ethanol solution
  18. Betadine
  19. Neosporin
  20. Saline

Equipment

  1. Pipette puller (Sutter Instruments, model: P97 )
  2. NalgeneTM 280 polyurethane tubing (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 8030-0060 )
  3. NalgeneTM 180 clear plastic PVC tubing (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 8000-0004 )
  4. Custom valve controller that delivers momentary pulses of 24 V with 5 watts power (modified pulse controller obtained from General Valve Corporation, model: 9-82-902 )
    Note: This product has been discontinued. As an alternative, use UltraMicroPumP III (World Precision Instruments, model: UMP3 ) with a TAXIC900 stereotaxic frame, which allows for a precise control of the rate of delivery of the virus.
  5. Air pressure regulator with gauge (General Cryogenics & Specialty Gas Co.)
  6. Hair clipper (e.g., Wahl Clipper, model: 09916-4301 )
  7. Heating pad, water-circulating
  8. Mouse stereotaxic apparatus (Stoeling, catalog number: 51730D )
  9. Solenoid valve (e.g., Parker Hannifin, part number: 001-0028-900 )
  10. Scalpel handle (Thomas Scientific, catalog number: 3883H10 )
  11. Camera (e.g., Microscope, model: 5MP , catalog number: AM7115MZT)
  12. Video monitor (e.g., Acer)
  13. Drill (Black & Decker, model: RTX )

Procedure

  1. Preparation for surgery (time 30-45 min)
    1. Pull the Drummond glass pipette using the Sutter pipette puller and then by stroking the tip on a Kimwipe break the tip back to a tip diameter between 20-40 μm (see Figure 1).
    2. Connect the solenoid pulser to the air pressure regulator with polyurethane tubing; connect the wall-mounted ball valve delivering air with polyethylene tubing (Figure 2).
    3. Backfill the glass pipette with virus solution (2 μl per mouse). Secure the pipette tip to the stereotaxic holder arm, and then connect the pipette using PVC tubing to a syringe attached to a 3-way stopcock valve. Put a drop of a virus solution (at least 2 μl per mouse) on a clean surface covered with paraffin to avoid breaking the pipette tip. Move the stereotaxic holder arm to touch the pipette tip to the drop of virus solution and then pull on the syringe to slowly apply negative pressure and fill the pipette with virus solution. Keep the pipette with the virus solution in a closed box, with wet paper towels in the bottom to maintain humidity, until you are ready to inject. Any surfaces in contact with the virus should be cleaned with 10% bleach solution.


      Figure 1. Images of the glass pipette used for the intracranial injection of the virus solution. Left, image of the pipette used for the AAV injection (graduated every 1 μl; maximum 10 μl). Right, photomicrograph of the pipette tip (33 μm diameter).


      Figure 2. Schematic of the setup using a solenoid valve to deliver time-controlled pulses of compressed air for the administration for the virus through a glass pipette

  2. Surgery and virus injection (time 20-30 min)
    1. Administer a subcutaneous injection in the neck of caprofen (5 mg/kg) pre-operatively for preemptive analgesia and for its anti-inflammatory actions.
    2. Anesthetize the mouse with a solution containing ketamine (100 mg/kg) and xylazine (10 mg/kg). If the mouse weighs less than 20 g, administer half the dose. Administer the drugs via intraperitoneal (i.p.) injection at volumes of 10 ml/kg.
    3. Shave the scalp with the hair clipper.
    4. Apply Puralube VET ointment to the eyes.
    5. Place the mouse on a heating pad (37 °C), secure the nose in the mouth bar and nose clamp and stabilize the head using the ear bars.
    6. Clean the skin above the skull, first with betadine and then with alcohol.
    7. Apply lidocaine solution using a Q-tip.
    8. Using a scalpel blade, make an incision and push the skin covering the skull to the side.
    9. Attach a piece of metal tubing to the stereotaxic probe holder and mark the skull using coordinates targeting the ventral tegmental area (VTA). For mice weighing more than 25 g use the following coordinates: anterior-posterior (AP) - 3.4 mm and lateral (L) -/+ 0.5 mm relative to bregma, and dorsoventral (DV) - 4.5 mm from the dura. Adjust the coordinates to AP - 3.0 mm and DV - 4.1 mm for mice weighing between 12 and 16 g, and to AP - 3.3 mm and DV - 4.3 mm for mice weighing between 17 and 24 g. Do not use the glass pipette to mark the coordinates on the skull, since the tip can easily break.
    10. Drill holes on both sides and clean the exposed skull with saline.
    11. Place the glass pipette with the virus solution in the probe holder of the stereotaxic apparatus and connect the pipette to the solenoid valve using PVC tubing (see schematic in Figure 1). Before lowering the pipette into the brain, make sure that the timed solenoid-controlled pulses of compressed air are efficiently ejecting the virus solution. If the virus solution is not coming out, gently clean the pipette tip using a Kimwipe saturated with saline.
    12. Gently pierce the dura with a needle before lowering the pipette.
    13. Slowly (approximately 0.2 mm per sec) lower the pipette to the target depth + 0.1 mm. Then raise the pipette 0.1 mm to the target depth (4.3 mm). This creates a pocket for the viral solution and reduces needed pressure when delivering the virus solution. Inject 1 μl of the virus solution per side using timed solenoid-controlled pulses of compressed air.
    14. Leave the pipette in place for 3 min after the injection to reduce back flux along the injection track, and then withdraw it.
    15. After both injections, clean the skull with saline, push the skin together with a forceps, and close the scalp incision with Vetbond, allow it to cure (1 min), and then apply Neosporin ointment. Then, remove the animal from the stereotaxic apparatus.
    16. Administer Carprofen daily for 3 days to alleviate pain.

Part II: Determining efficacy and selectivity of the ChR2 expression in dopamine neurons using immunofluorescence

Viral transfection delivers multiple copies of ChR2 and induces strong expression. However, ChR2 expression varies among animals so the specificity and efficacy of the transfection needs to be determined and taken into account when determining a functional connectome. Visualization of immunostaining of brains sections for the EYFP tag enables tracing dopaminergic projections, assessing their dopaminergic status and by guiding recordings provides a comprehensive basis for systematic recordings.

Materials and Reagents

  1. Multi-well plates (Thomas Scientific, catalog number: 1219C16 )
  2. Petri dishes (Thomas Scientific, catalog number: 1182N84 )
  3. Coverslips (e.g., Corning, catalog number: 2980-245 )
  4. Microscope slides coated (e.g., Thomas Scientific, catalog number: 1178T40 )
  5. Ketamine HCl (KetaVed, Vedco)
  6. Xylazine (Akorn, AnaSed®)
  7. Heparin (Swiss Vault Engine, catalog number: 25021-400-66 )
  8. Phosphate buffer (Sigma-Aldrich, catalog number: P3619 )
  9. Dulbecco’s phosphate buffer saline (DPBS) (Sigma-Aldrich, catalog number: D5652 )
  10. Glycine (molecular weight: 75.07) (Sigma-Aldrich, catalog number: 410225 )
  11. Normal donkey serum (EMD Millipore, catalog number: S30-100mL )
  12. Triton (Sigma-Aldrich, catalog number: X100 )
  13. Rabbit polyclonal antibody directed against green fluorescence protein (GFP) (EMD Millipore, catalog number: AB3080 ), which recognizes enhanced yellow fluorescence protein (EYFP)
  14. Mouse monoclonal antibody against tyrosine hydroxylase (TH) (EMD Millipore, catalog number: MAB318 ), which recognizes a specific marker of dopamine neurons
  15. Donkey anti-rabbit Alexa Fluor® 488 secondary antibody (Thermo Fisher Scientific, Invitrogen, catalog number: A-21206 )
  16. Donkey anti-mouse Alexa Fluor® 594 secondary antibody (Thermo Fisher Scientific, Invitrogen, catalog number: A-21203 )
  17. Prolong Gold mounting medium (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: P36930 )
  18. Paraformaldehyde (PFA, 16%) (Electron Microscopy Sciences, catalog number: 15710 )
  19. Glycerol (molecular weight: 92.09) (Sigma-Aldrich, catalog number: G5516 )
  20. Ethylene glycol (molecular weight: 62.07) (Sigma-Aldrich, catalog number: 324558 )
  21. Tris HCl (Sigma-Aldrich, catalog number: T2413 )

Equipment

  1. Brush (small paint brush)
  2. Vibrating microtome (Leica Biosystems Nussloch, model: VT1200S )
  3. Laboratory shaker (Benchmark Scientific, model: BlotBoy )
  4. Slide storage boxes (Thomas Scientific, catalog number: 1202N78 )
  5. Confocal scanning microscope (Olympus, model: Fluoview FV1000 )

Software

  1. ImageJ (imagej.nih.gov/ij/download.html) (Abràmoff et al., 2004)

Procedure

  1. Brain fixation and slicing (1 day)
    1. After 3-5 weeks post injection, anesthetize mouse with an i.p. injection of a ketamine (100 mg/kg)-xylazine (15 mg/kg) solution.
    2. Rapidly perfuse intracardially with 1 ml of warm phosphate buffer (30 °C, 0.1 M, pH 7.4) containing 10,000 IU heparin/L, followed by 5 ml of cold phosphate buffer and then by 5 ml of 4% PFA in phosphate buffer.
    3. Remove the brain and postfix in 4% PFA solution overnight.
    4. Fill each well of the multi-well plate with 1 ml of cryoprotectant solution.
    5. Slice the brain on the vibrating microtome at 50 μm to generate coronal sections.
    6. Collect sections into the multi-well plate.
    7. Keep brain sections at -20 °C until processing.

  2. Immunofluorescence staining and imaging (3 days)
    1. Wash the sections in the multi-well plate with PBS for 5 min in a shaker (12 rpm); repeat this twice.
    2. Move the sections to a new multi-well plate with glycine (100 mM; 1 ml per well) to quench aldehydes, and put the multi-well plate on the shaker for 30 min.
    3. Wash sections in PBS for 5 min; repeat this twice.
    4. Block sections with normal donkey serum (10%) diluted in PBS with 0.1% Triton for 2 h in a shaker. Add 1 ml of this blocking solution per well.
    5. Dilute the antibodies, anti-GFP (dilution: 1:2,000) and anti-TH (dilution: 1:5,000), in PBS with 2% of normal donkey serum and 0.02% of Triton.
    6. Move the sections to a new multi-well plate containing both the anti-GFP and anti-TH antibody (1 ml per well), and let the sections incubate on a shaker at 4 °C overnight (minimum 16 h; for better results, leave the antibody incubating over the weekend).
    7. Wash the sections in PBS for 5 min on the shaker. Repeat this twice.
    8. Incubate for 2 h with anti-rabbit and anti-mouse secondary antibodies (dilution: 1:200 in PBS-T 0.02%) on the shaker at room temperature.
    9. Wash the sections in PBS for 5 min; repeat this twice.
    10. Mount sections on slides in phosphate buffer and let them dry for 5 min.
    11. When completely dried, put a few drops of the mounting medium on the section and coverslip, being careful not to create bubbles.
    12. Leave the slides in the dark, overnight at room temperature.
    13. Apply nail polish around the edges of the coverslip; this is to prevent the coverslip from moving as the mounting medium will not dry.
    14. Store slides in slide storage boxes at 4 °C. The fluorescence signal typically lasts for 2 years.
    15. Using a confocal scanning microscope to image the region of interest, in this case the ventral tegmental area and substantia nigra pars compacta (see Figure 1 in Mingote et al., 2015). Use a mouse atlas to make sure that the same regions are imaged for all subjects.
    16. Take confocal photomicrograph stacks (60x oil objective; optical zoom 1.4x, and z-step increment 0.42 μm; 800 x 800 pixels image frames with a pixel size of 0.189 μm2) through the entire tissue section (40 to 60 images).

Data analysis

  1. Use ImageJ to adjust for contrast and brightness of the confocal stacks linearly.
  2. Make a z-projected image of the confocal stacks (this image will be used to count) but keep the image with all the confocal stacks also open (this image will be used to identify the different types of cells).
  3. Identify the number of TH positive (+)/ChR2-EYFP (-), TH (-)/ChR2-EYFP (+) and TH (+)/ChR2-EYFP (+) cells in each image composed of several confocal stacks by scrolling up and down the z-axis and comparing images in the red and green channels.
  4. Count the number of each type of cell in the z-projected image using the ImageJ cell-counter plug-in.
  5. Calculate efficacy and specificity percentage values for each stereotaxic position. The efficacy of the virus transfection is calculated as the number of TH (+) and ChR2-EYFP (+) in relation to the total number of TH (+) cells. The specificity of the virus transfection is calculated as the number of TH (+) and ChR2-EYFP (+) in relation to the total number of ChR2 (+) cells (see Figure 3 below, or Figure 2 in Mingote et al., 2015).
  6. Differences along the anterior-posterior and medial-lateral axes are analyzed using the non-parametric Friedman’s test, with significance set at 0.05.
  7. Identify the projection areas in other sections of the cells of interest, in this case the ventral midbrain dopamine neurons, which determine the areas to record from.
     

    Figure 3. Determining the efficacy and specificity of the ChR2 expression in dopamine neurons. Top panels, confocal z-projected photomicrographs from the lateral VTA and SNc showing cells expressing tyrosine hydroxylase (TH) in magenta, ChR2-EYFP in green, and cells expressing both markers in white. In the panels on the left, white stars indicate TH (+) and ChR2-EYFP (+); blue stars indicate TH (+) and ChR2 (-) cells. Bottom left, Schematic of the ventral midbrain showing the locations where the photomicrographs were taken relative to the AAV injection site. Bottom right, efficacy and specificity graphs based on the cell counts done in the photomicrographs on the top. Efficacy decreases in dopamine neurons located further from the injection site. The specificity using AAV-ChR2-EYFP is very high; the number of TH (-) and ChR2-EYFP- cells is negligible (none are seen here).

Notes

  1. It has been reported that IgG is present in microglia and this leads to spurious labeling with anti-mouse secondary antibodies; however it has also been shown that such labeling is mouse strain-specific and depends on the immunostaining procedure used (Hazama et al., 2005). Our immunostaining protocol did not produce any non-specific staining of microglia. Alternatively, anti-TH antibody made in chicken or rabbit could be used.
  2. ChR2 can be expressed in dopamine neurons transgenically, using Ai32 mice (B6;129S-Gt(ROSA)26Sortm32(CAG-COP4*H134R/EYFP)Hze/J;RRID:IMSR_JAX:012569) (Madisen et al., 2012). The transgenic strategy yields reliable expression from animal to animal that cannot be obtained with viral injections (Figure 4). For direct comparison of these two approaches to target dopamine neurons see also Tritsch et al. (2012, supplemental Figure 1). However, the ChR2 specificity in transgenic strategies can be compromised due to ectopic expression. For example, using the serotonin transporter to drive cre expression and ChR2 transgenically (i.e., SERT-cre::Ai32 mice) would not be a good strategy to target Raphe nucleus serotonin neurons selectively since the SERT promotor is transiently active in thalamic cells during development (Narboux-Nême et al., 2008), and those cells would continue to express ChR2 in adulthood.


    Figure 4. Expression of ChR2-EYFP in TH (+) neurons in the ventral midbrain of DATIREScre mice, induced either by a bilateral injection of an AAV-ChR2-EYFP (top) or breeding with Ai32 mouse (bottom). With both strategies, ChR2 expression is specific to TH (+) cells. However, the viral strategy may not target all the TH (+), as shown on the left side of the photomicrographs in the top panel. When using the viral strategy, ChR2 expression efficacy is highly dependent on the success of the injection and the spread of the virus within the brain region of interest for each injected mouse. For this reason, the efficiency and reliability of expression is much higher with the transgenic strategy (bottom panel). Scale bar represents 400 μm.

Recipes

  1. 4% paraformaldehyde (PFA) solution (20 ml)
    10 ml of 0.2 M phosphate buffer
    5 ml of ddH2O
    5 ml of 16% PFA
  2. Cryoprotectant solution (100 ml)
    30 ml of glycerol
    30 ml ethylene glycol
    60 ml 0.1 M Tris HCl

Part III: Determining the excitatory functional connectome of dopamine neurons using slice electrophysiology and optogenetics

Materials and Reagents

  1. Nalgene 4 mm syringe filter, 0.2 µm pore size, cellulose acetate membrane (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 171-0020 )
  2. 1 ml Tuberculin slip tip syringe, 1 ml (BD, catalog number: 309659 )
  3. Nonmetallic syringe needle for filling micropipettes, 28 G, 67 mm (World Precision Instruments, catalog number: MF28G67-5 )
  4. Pipette tips  
  5. Glass pipette
  6. Ketamine HCl (KetaVed, Vedco)
  7. Xylazine (Akorn, AnaSed®)
  8. Sodium chloride (NaCl) (molecular weight: 58.44) (Sigma-Aldrich, catalog number: 746398 )
  9. Potassium chloride (KCl) (molecular weight: 74.55) (Sigma-Aldrich, catalog number: 746436 )
  10. Sodium bicarbonate (NaHCO3) (molecular weight: 84.01) (Sigma-Aldrich, catalog number: S6014 )
  11. Sodium phosphate monobasic (NaH2PO4) (molecular weight: 119.98) (Sigma-Aldrich, catalog number: S3139 )
  12. Calcium chloride dehydrate (CaCl2) (molecular weight: 147.01) (Sigma-Aldrich, catalog number: 223506 )
  13. Magnesium chloride (MgCl2) (molecular weight: 95.21) (Sigma-Aldrich, catalog number: M8266 )
  14. Glucose (molecular weight: 180.16) (Sigma-Aldrich, catalog number: G6152 )
  15. Carbogen (95% O2 + 5% CO2)
  16. SR95531 (gabazine, GABAA antagonist) (molecular weight: 368.23) (Abcam, catalog number: Asc-042 )
  17. CGP55845 (GABAB antagonist) (molecular weight: 438.71) (Tocris Bioscience, catalog number: 1248 )
  18. SCH23390 (D1 antagonist) (molecular weight: 324.24) (Tocris Bioscience, catalog number: 0925 )
  19. (−)-Sulpiride (D2 antagonist) (molecular weight: 341.42) (Tocris Bioscience, catalog number: 0895 )
  20. Scopolamine hydrobromide (muscarinic antagonist) (molecular weight: 384.27) (Tocris Bioscience, catalog number: 1414 )
  21. Lidocaine N-ethyl bromide (QX-314, intracellular sodium channel blocker) (molecular weight: 343.30) (Sigma-Aldrich, catalog number: L5783 )
  22. 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt (CNQX) (molecular weight: 276.12) (Tocris Bioscience, catalog number: 1045 )
  23. Gluconic acid (molecular weight: 196.16) (Sigma-Aldrich, catalog number: G1951 )
  24. Cesium hydroxide hydrate (CsOH·H2O) (molecular weight: 149.91) (Sigma-Aldrich, catalog number: C8518 )
  25. HEPES (molecular weight: 238.30) (Sigma-Aldrich, catalog number: RDD002 )
  26. EGTA (molecular weight: 380.35) (Sigma-Aldrich, catalog number: E3889 )
  27. ATP-Na2 (molecular weight: 551.14) (Sigma-Aldrich, catalog number: A2383 )
  28. GTP-Na2 (molecular weight: 523.18) (Sigma-Aldrich, catalog number: G8877 )
  29. High-glucose aCSF (see Recipes)
  30. Standard aCSF (see Recipes)
  31. Intracellular solution (see Recipes)

Equipment

  1. Scissors
  2. Vibrating microtome (Leica Biosystems Nussloch, model: VT1200S )
    Note: Different vibratomes are used for histology and for slice recording to avoid detrimental effects of formaldehyde on slice health.
  3. Pipette puller (Sutter Instruments, model: P97 )
  4. Patch pipettes, standard borosilicate glass capillaries (OD/ID 1.5/0.84 mm) with filament (World Precision Instruments, catalog number: 1B150F-4 )
  5. CCD camera (Olympus, model: OLY-150 , discontinued), as alternative, scientific CMOS camera with live imaging mode (e.g., Hamamatsu Photonics, model: C11440-42U ) can be used
  6. High-Power blue LED (470 nm; LED controller current set at 1 A, corresponding to a LED voltage of 10 V) (Thorlabs, model: DC4100 )
  7. Fluorescence microscope (Olympus, model: BX61WI ) with a 60x water-immersion lens
  8. Recording chamber (perfusion chamber round) (Siskiyou, model: PC-R )
  9. Reference electrode (Ag-AgCl electrode, 1 mm diameter, 3 mm long) (World Precision Instruments, catalog number: EP1 )
  10. Micromanipulator (mechanical, Huxley-style) (Siskiyou, model: MX310R )
  11. Custom made ‘harp’ to secure slices to recording chamber (U-shaped flattened platinum-iridium wire with single nylon strings attached by cyanoacrylate glue)
  12. Temperature controller of bath solution (Warner Instruments, model: TC 344B )
  13. Patch clamp amplifier (Molecular Devices, model: Axopatch 200B )
  14. Computer for data acquisition with interface (HEKA Elektronik Dr. Schulze, model: InstruTECH ITC-18 ) or (Molecular Devices, model: Axon DigiData 1500 )

Software

  1. Axograph X (Axograph Scientific) or pClamp 10 (Molecular Devices)

Procedure

  1. Preparation for slice recording (1 day)
    1. High-glucose artificial cerebrospinal fluid (aCSF) is used when incubating brain slices; standard aCSF is used for patch-clamp recordings. For both, prepare a 10x stock solutions (see Recipes section).
    2. Prepare Cesium-gluconate with QX-314 intracellular solution (see Recipes section) and aliquot.

  2. Making brain slices and patch-clamp recording (1 day)
    1. At 3-5 weeks post injection, anesthetize the mouse with ketamine (100 mg/kg) + xylazine (15 mg/kg).
    2. Open the skull along the sagittal suture with scissors, reflect the skull flaps, and remove the brain into ice-cold high-glucose aCSF (75 mM NaCl, 2.5 mM KCl, 26 mM NaHCO3, 1.25 mM NaH2PO4, 0.7 mM CaCl2, 2 mM MgCl2 and 100 mM glucose; see Recipes section), saturated with carbogen.
    3. Make 300 μm thick slices for each area of interest. To establish the functional connectome of the VTA dopamine neurons, recordings were done in four different coronal sections containing the striatum, the amygdala, the hippocampal formation, or the anterior cortex.
    4. Preincubate slices in high-glucose aCSF saturated with carbogen for at least 1 h at room temperature for recovery.
    5. During the incubation time, pull patch pipettes with pipette resistance between 4 and 8 MΩ (tip diameter ~1 µm).
    6. Transfer one slice to the recording chamber (submerged, 500 μl volume) on the stage of the fluorescence microscope and secure the slice with the harp. The recording chamber should be continuously perfused (1.5 ml/min) with standard aCSF (125 mM NaCl, 2.5 mM KCl, 25 mM NaHCO3, 1.25 mM NaH2PO4, 2 mM CaCl2, 1 mM MgCl2 and 25 mM glucose; see Recipes section) saturated with carbogen, and maintained at 31-33 °C.
    7. Glutamatergic responses are isolated by blocking dopamine, GABA and acetylcholine actions with a cocktail of antagonists added to the perfusate: 10 μM SR95531, 3 μM CGP55345, 10 μM SCH23390, 10 μM (−)-sulpiride and 1 μM scopolamine.
    8. Focus on the area of interest using a 10x objective and then use the 60x and a camera to identify the cell to patch. All recordings should be done in areas with visible ChR2-EYFP axons.
    9. Voltage-clamp recordings are performed with a -75 mV holding potential with the sodium channel blocker (QX-314) in the intracellular solution to block active currents. Cell activity can be monitored in cell attached mode. Once whole-cell mode is achieved, neurons can only be identified based on cell body location, size, and shape, since cesium-based pipette solution including QX-314 blocks intrinsic activity.
    10. After locating a healthy cell, backfill the patch pipette with the filtered (by 0.2 µm pore syringe filter) cesium-gluconate intracellular solution using the non-metal syringe needle, and mount the pipette in the pipette holder on the micromanipulator. Before immersing the pipette tip in the bath, apply a gentle positive pressure to the glass pipette; this prevents dilution of the intracellular solution due to aspiration of bath solution and clogging of the pipette.
    11. Advance the pipette to the chosen cell with the micromanipulator. After touching the cell membrane with the tip of the pipette, gently advance to dimple the membrane. Release the positive pressure and confirm that the tip resistance increases, then apply gentle negative pressure to the pipette and apply negative holding potential to achieve a GΩ seal, wait 1-2 min for the seal to maximize. Apply further negative pressure to rupture the membrane at the tip of the pipette for whole cell recording, quickly releasing the negative pressure from the pipette after breaking through.
    12. To minimize baseline noise for better detection of small amplitude excitatory postsynaptic currents (EPSCs) series resistance is not compensated (see Notes).
    13. Data acquisition using Axograph X or pClamp commences 5 min after achieving whole-cell mode, to allow for diffusion of intracellular solution into recorded cells.
    14. Photostimulate with short flashes (5 msec at 0.1 Hz) of a high-power blue LED illumination every 10 sec (0.1 Hz) to evoke synaptic responses. To monitor series resistance, a 5 mV, 5 msec voltage pulse is applied 50 msec before each photostimulation. Record a minimum of 20 responses per cell.
    15. Glutamatergic responses should be confirmed by perfusion of 40 μM CNQX, allowing at least 5 min after drug application for full effect.

Data analysis

  1. For each cell, average 10 consecutive traces. Discard the first trace, since the EPSC amplitude is always significantly larger due to the rested state of the synaptic connection.
  2. Measure the peak amplitude within the 50 msec post-photostimulation time window.
  3. The detection threshold for determination of connections should be set to the mean plus 2 standard deviations of the baseline (including spontaneous EPSCs) amplitude in the 100 msec time window preceding the photostimulation. When the peak amplitude of post-photostimulation EPSCs exceeds this threshold, the cell is counted as having a dopamine neuron glutamatergic connection (see Figure 5).


    Figure 5. Determining the strength and incidence of the dopamine neuron glutamatergic connections. A-C. Traces showing examples of a cell recorded with no connection (pyramidal cell 1) and two cells with connections of different strengths (pyramidal cell 2 and 3; blue traces). The AMPA/kainate-receptor antagonist CNQX (40 M) completely blocked the photostimulated EPSC (pyramidal cell 3; red trace) showing glutamate mediation. Gray traces were used to calculate the mean amplitude of the baseline plus 2 standard deviations (SD); responses that exceeded this threshold were considered to show connections. D. Graph on the bottom right, is a plot of the data shown in this figure. The strength of the connection is calculated as the mean amplitude of the responses in the two cells with a connection, while the incidence of connections is the percentage of cells that show a connection relative to all cells recorded, in this case, 2 out of 3 cells. Recordings shown are from the entorhinal cortex. Photostimulation is indicated by the blue bar.

Notes

  1. The bath and pipette solutions generate a ~15 mV liquid junction potential. To correct junction potentials online and to determine the -75 mV holding, set holding command of amplifier to -60 mV (Neher, 1992).
  2. Since series resistance compensation uses a positive feedback circuit, online compensation increases baseline noise. Generally, if a response is small and slower, series resistance compensation does not affect the amplitude or time course of the response, which we confirmed. However, series resistance compensation should be considered for recordings of larger and faster responses, online or offline (with analysis software).
  3. To improve the identification of the postsynaptic cell, it is possible to genetically target the recorded cell with GFP or fill the recording cell with a fixable marker (e.g., biocytin, Alexa fluorescent dyes) and then fix the tissue for double labeling of the cell with a known cell marker.

Recipes

  1. 10x aCSF stock solution without glucose, magnesium or calcium
    43.83 g of NaCl
    1.86 g of KCl
    21.84 g of NaHCO3
    1.5 g of NaH2PO4
    Add ddH2O to make 1 L solution and store it at 4 °C
  2. High-glucose aCSF (1 L)
    On the day of the slice recording experiment make the high-glucose aCSF
    Dilute the 10x stock solution 10 times (100 ml of the stock solution in 900 ml of ddH2O)
    Add 18.02 g of glucose
    Add 2 ml of a 1 M solution of MgCl2 (add 95.21 mg per 1 ml ddH2O)
    Add 0.7 ml of a 1 M solution of CaCl2 (add 147 mg per 1 ml ddH2O)
  3. Standard aCSF (1 L)
    On the day of the slice recording experiment make the standard aCSF
    Dilute the stock solution 10 times (100 ml of the stock solution in 900 ml of ddH2O)
    Add 4.5 g of glucose
    Add 1 ml of a 1 M solution of MgCl2 (add 95.21 mg per 1 ml ddH2O)
    Add 2 ml of a 1 M solution of CaCl2 (add 147 mg per 1 ml ddH2O)
    If adding CaCl2 causes precipitation of calcium carbonate, saturate the solution with carbogen to clear the precipitation
  4. Intracellular solution (Cesium-gluconate containing QX-314, 25 ml), Osmolarity 287 mOsm
    1 ml of the 3.25 M gluconic acid (molarity of the Sigma-Aldrich solution)
    3.25 ml of 1 M CsOH solution (add 130 mg per 1 ml of ddH2O)
    50 μl of 1 M MgCl2 solution (add 95.21 mg per 1 ml ddH2O)
    2.5 μl of 1 M CaCl2 solution (add 147 mg per 1 ml ddH2O)
    2.5 ml of 50 mM QX-314 solution (add 17.2 mg per 1 ml ddH2O)
    250 μl of 1 M HEPES solution (add 238.30 mg per 1 ml ddH2O)
    9.5 mg of EGTA
    Add 17.948 ml of ddH2O
    Let the solution sit overnight at 4 °C to stabilize the pH
    Adjust the pH to 7.3 with CsOH solution
    Add 25.4 mg of ATP-Na2
    Add 3.9 mg of GTP-Na2
    Filter the solution with a 2 μm filter
    Make 1 ml aliquots and store them at -20 °C

Acknowledgments

This protocol was used to obtain the data published in the Journal of Neuroscience (Mingote, S., Chuhma, N., Kusnoor, S. V., Field, B., Deutch, A. Y. and Rayport, S. [2015]). Functional connectome analysis of dopamine neuron glutamatergic connections in forebrain regions. J Neurosci 35[49]: 16259-16271). Procedures involving mice were conducted in accordance with the guidelines of the National Institute of Health Guide for the Care and Use of Laboratory Animals under protocols approved by the Institutional Animal Care and Use Committees of Columbia University and New York State Psychiatric Institute. The work was supported by a NARSAD Young Investigator award (SM), DA017978 and MH087758 (SR).

References

  1. Abràmoff, M. D., Magalhães, P. J. and Ram, S. J. (2004). Image processing with ImageJ. Biophotonics international 11(7): 36-42.
  2. Callaway, E. M. and Luo, L. (2015). Monosynaptic circuit tracing with glycoprotein-deleted rabies viruses. J Neurosci 35(24): 8979-8985.
  3. Chuhma, N. (2015). Optogenetic analysis of striatal connections to determine functional connectomes. In: Hiromu, Y. (Ed.). Optogenetics. Springer pp: 265-277.
  4. Chuhma, N., Tanaka, K. F., Hen, R. and Rayport, S. (2011). Functional connectome of the striatal medium spiny neuron. J Neurosci 31(4): 1183-1192.
  5. Faget, L., Osakada, F., Duan, J., Ressler, R., Johnson, A. B., Proudfoot, J. A., Yoo, J. H., Callaway, E. M. and Hnasko, T. S. (2016). Afferent inputs to neurotransmitter-defined cell types in the ventral tegmental area. Cell Rep 15(12): 2796-2808.
  6. Hazama, G. I., Yasuhara, O., Morita, H., Aimi, Y., Tooyama, I. and Kimura, H. (2005). Mouse brain IgG-like immunoreactivity: strain-specific occurrence in microglia and biochemical identification of IgG. J Comp Neurol 492(2): 234-249.
  7. Hnasko, T. S., Hjelmstad, G. O., Fields, H. L. and Edwards, R. H. (2012). Ventral tegmental area glutamate neurons: electrophysiological properties and projections. J Neurosci 32(43): 15076-15085.
  8. Madisen, L., Mao, T., Koch, H., Zhuo, J. M., Berenyi, A., Fujisawa, S., Hsu, Y. W., Garcia, A. J., 3rd, Gu, X., Zanella, S., Kidney, J., Gu, H., Mao, Y., Hooks, B. M., Boyden, E. S., Buzsaki, G., Ramirez, J. M., Jones, A. R., Svoboda, K., Han, X., Turner, E. E. and Zeng, H. (2012). A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing. Nat Neurosci 15(5): 793-802.
  9. Mingote, S., Chuhma, N., Kusnoor, S. V., Field, B., Deutch, A. Y. and Rayport, S. (2015). Functional connectome analysis of dopamine neuron glutamatergic connections in forebrain regions. J Neurosci 35(49): 16259-16271.
  10. Narboux-Nême, N., Pavone, L. M., Avallone, L., Zhuang, X. and Gaspar, P. (2008). Serotonin transporter transgenic (SERTcre) mouse line reveals developmental targets of serotonin specific reuptake inhibitors (SSRIs). Neuropharmacology 55(6): 994-1005.
  11. Neher, E. (1992). Correction for liquid junction potentials in patch clamp experiments. Methods Enzymol 207: 123-131.
  12. Root, D. H., Mejias-Aponte, C. A., Zhang, S., Wang, H. L., Hoffman, A. F., Lupica, C. R. and Morales, M. (2014). Single rodent mesohabenular axons release glutamate and GABA. Nat Neurosci 17(11): 1543-1551.
  13. Tritsch, N. X., Ding, J. B. and Sabatini, B. L. (2012). Dopaminergic neurons inhibit striatal output through non-canonical release of GABA. Nature 490(7419): 262-266.

简介

神经回路中的功能连通性由其连接的强度,发病率和神经递质特性决定(Chuhma,2015)。使用光遗传学,可以系统地测量识别的神经元群体和定义的突触后靶神经元之间的功能性突触连接,以便确定该识别群体的功能性连接群。这里我们描述了用于研究由谷氨酸共转播介导的腹侧中脑多巴胺神经元的兴奋性功能性连接体的实验方案(Mingote等,2015)。通过将编码通道视紫红质(ChR2)的腺相关病毒(AAV)注射到DATIREScre小鼠的腹侧中脑中,使多巴胺神经元变得光敏。多巴胺合成酶酪氨酸羟化酶的免疫荧光证实ChR2表达在多巴胺神经元中的功效和特异性。然后,切片膜片钳记录由接受多巴胺神经元投影的区域中的神经元产生,并且确定兴奋性连接的发生率和强度。所有接受多巴胺神经元投射的区域的连接发生率和强度的总结构成功能性连接体。
【背景】为了建立特定神经回路的功能,有必要确定解剖连接,解剖连接的映射及其功能连接,连接的强度,发病率和神经递质性质的映射。使用单因素限制的病毒性突触后追踪技术,可以描述包括多巴胺系统在内的神经回路的复杂解剖连接(Callaway and Luo,2015; Faget等,2016)。由于轴突的混合使得选择性电刺激不可能,因此这些电路的功能连通性更难确定。随着光遗传学的出现,有可能选择性地刺激遗传定义的细胞群体。这允许鉴定由纹状体中等多刺神经元(Chuhma等,2011),腹侧中脑谷氨酸神经元(Hnasko等,2012; Root等,2014)和多巴胺/谷氨酸神经元(Mingote et al。,2011)等等,2015)。此外,以系统和全面的方式使用光遗传学将特定神经元群体的连接的发生率和强度映射到定义的突触后靶区域,确定了定义的神经元群体的功能性连接(Chuhma et al。,2011; Mingote et al。,2015 )。在这个协议中,我们描述了如何确定任何遗传定义的神经元群体的功能连接。例如,我们专注于由谷氨酸共转播介导的多巴胺神经元的兴奋性功能性连接(Mingote等,2015)。

关键字:通道视紫红质, 多巴胺, 共同传递, 膜片钳, 免疫荧光, 腺相关病毒

第一部分:通过病毒转染诱导多巴胺神经元中的channelrhodopsin表达 >

材料和试剂

  1. 玻璃PCR微量移液管,1μl标记(Drummond Scientific,目录号:5-000-1001-X10)
  2. Kimwipe
  3. 注射器
  4. Q-tip
  5. 手术刀片No.11(Thomas Scientific,目录号:3883B59)

  6. 小鼠(The JACKSON LABORATORY,Strain 006660:DAT IREScre
    注意:该敲入小鼠在内源性多巴胺转运蛋白(DAT)启动子的转录控制下表达cre重组酶。为了最小化对DAT启动子功能的干扰,cre重组酶表达通过内部核糖体进入序列(IRES)从3'非翻译区驱动。
  7. 使用AAV5-EF1α-DIO-hChR2(H134R)-EYFP(滴度:8×10e-12病毒体/ml)(Addgene,目录号:20298)来驱动ChR2-EYFP的依赖性表达。腺相关病毒(AAV)可以从北卡罗来纳大学的载体核心的Karl Deisseroth博士的MTA获得;该AAV是血清型5和复制无能。
  8. 石蜡
  9. 10%漂白溶液
  10. Carprofen(Rimadyl,Zoetis)
  11. 盐酸氯胺酮(Ketved,Vedco)
  12. 赛拉嗪(Akorn,AnaSed ®
  13. Puralube VET软膏,无菌眼部润滑剂(Dechra)
  14. 盐酸利多卡因(40 mg/ml)用于局部麻醉(Boehringer Ingelheim)
  15. Vetbond,氰基丙烯酸正丁酯胶粘剂(3M)
  16. 70%乙醇溶液
  17. Betadine
  18. 新孢子素
  19. 盐水

设备

  1. 移液器拉杆(Sutter Instruments,型号:P97)
  2. Nalgene TM聚氨酯管(Thermo Fisher Scientific,Thermo Scientific TM,目录号:8030-0060)
  3. Nalgene TM 180透明塑料PVC管(Thermo Fisher Scientific,Thermo Scientific TM,目录号:8000-0004)
  4. 定制阀控制器,提供24 V的瞬时脉冲,功率为5瓦(通用阀门公司获得的改进的脉冲控制器,型号:9-82-902)
    注意:本产品已停产。作为替代方案,使用具有TAXIC900立体定位框架的UltraMicroPumP III(世界精密仪器,型号:UMP3),可以精确控制病毒的传播速率。
  5. 空气压力调节器(普通冷冻和特种气体公司)
  6. 吹风机(例如,,Wahl Clipper,型号:09916-4301)
  7. 加热垫,水循环
  8. 鼠标立体定位仪(Stoeling,目录号:51730D)
  9. 电磁阀(例如,Parker Hannifin,部件号:001-0028-900)
  10. 手术刀柄(Thomas Scientific,目录号:3883H10)
  11. 相机(例如,,显微镜,型号:5MP,目录号:AM7115MZT)
  12. 视频监视器(例如,,宏碁)
  13. 钻(Black& Decker,型号:RTX)

程序

  1. 手术准备(时间30-45分钟)
    1. 使用Sutter吸管拔出器拉出Drummond玻璃移液器,然后通过抚摸Kimwipe上的尖端将尖端回到20-40μm之间的尖端直径(见图1)。
    2. 使用聚氨酯管将电磁脉冲发生器连接到气压调节器;连接带有聚乙烯管道的壁挂式球阀(图2)
    3. 用病毒溶液回填玻璃移液管(每只鼠2μl)。将移液管尖固定到立体定位支架臂上,然后使用PVC管将移液管连接到连接到三通旋塞阀上的注射器。将一滴病毒溶液(每只小鼠至少2μl)放在用石蜡包裹的干净的表面上,以免打破移液器吸头。移动立体定位支架臂以将移液器尖端接触病毒液滴,然后拉动注射器以缓慢施加负压,并用病毒溶液填充移液管。将吸管与病毒溶液保持在一个封闭的盒子里面,湿纸巾在底部保持湿度,直到你准备好注射。任何与病毒接触的表面均应用10%漂白溶液清洗。


      图1.用于颅内注射病毒溶液的玻璃移液管的图像。左侧,用于AAV注射的移液管图像(每1μl分级;最大10μl)。右,移液管尖端的显微照片(直径33μm)。


      图2.使用电磁阀提供时间控制脉冲的压缩空气用于通过玻璃移液器管理病毒的设置示意图

  2. 手术和病毒注射(时间20-30分钟)
    1. 管理皮下注射在卡洛芬颈部(5 mg/kg)术前用于抢先镇痛及其抗炎作用。
    2. 用含有氯胺酮(100mg/kg)和赛拉嗪(10mg/kg)的溶液麻醉小鼠。如果小鼠体重小于20g,则服用一半剂量。以10ml/kg的体积通过腹膜内(i.p.)注射给药。
    3. 用剪发器刮头皮。
    4. 将Puralube VET软膏用于眼睛。
    5. 将鼠标放在加热垫(37°C)上,将鼻子固定在嘴巴和鼻夹中,并使用耳塞稳定头部。
    6. 清洁头骨上方的皮肤,先用betadine然后用酒精。
    7. 使用Q-tip使用利多卡因溶液。
    8. 使用手术刀刀片,做一个切口,将覆盖颅骨的皮肤推到侧面。
    9. 将一块金属管连接到立体定位探头支架上,并使用坐标指向腹侧被盖区域(VTA)标记颅骨。对于体重超过25g的小鼠,使用以下坐标:前后(AP) - 3.4mm和相对于胎膜的外侧(L) -/+ 0.5mm,以及距硬脑膜的背部(DV) - 4.5mm。将体重为12至16g的小鼠的坐标调整为AP-3.0mm和DV-4.1mm,对于体重为17至24g的小鼠,将坐标调整为AP-3.3mm和DV-4.3mm。不要使用玻璃移液管标记颅骨上的坐标,因为尖端可以轻易破裂。
    10. 在两侧钻孔,用盐水清洁暴露的头骨。
    11. 将带有病毒溶液的玻璃移液管置于立体定位设备的探头支架中,并使用PVC管将移液器连接到电磁阀(参见图1中的原理图)。在将移液器放入大脑之前,请确保压缩空气的定时电磁控制脉冲有效地喷射病毒溶液。如果病毒解决方案没有出来,使用饱和盐水的Kimwipe轻轻清洁移液器吸头。
    12. 在放下移液管之前,用针头轻轻地刺穿硬膜。
    13. 缓慢(每秒约0.2 mm)将移液管降低至目标深度+ 0.1 mm。然后将移液管提升0.1 mm至目标深度(4.3 mm)。这为病毒解决方案创造了一个口袋,并在传送病毒解决方案时减少了所需的压力。使用定时电磁阀控制的压缩空气脉冲每侧注入1μl病毒溶液。
    14. 注射后放置移液器3分钟,以减少注射轨道的回流,然后将其取出。
    15. 两次注射后,用盐水清洗头骨,用镊子将皮肤推入皮肤,并用Vetbond将头皮切口封闭,使其固化(1分钟),然后应用新孢菌素软膏。然后,从立体定位装置中取出动物。
    16. 每天处理Carprofen 3天以减轻疼痛。

第二部分:使用免疫荧光测定ChR2表达在多巴胺神经元中的功效和选择性 >

病毒转染可提供多拷贝的ChR2并诱导强烈的表达。然而,ChR2表达因动物而异,因此在确定功能性连接物时需要确定转基因的特异性和功效。用于EYFP标签的脑部分的免疫染色的可视化使得能够追踪多巴胺能投射,评估其多巴胺能状态并通过指导记录为系统记录提供了综合的基础。

材料和试剂

  1. 多孔板(Thomas Scientific,目录号:1219C16)
  2. 培养皿(Thomas Scientific,目录号:1182N84)
  3. Coverslips(例如,,Corning,目录号:2980-245)
  4. 显微镜载玻片(例如,Thomas Scientific,目录号:1178T40)
  5. 盐酸氯胺酮(Ketved,Vedco)
  6. 赛拉嗪(Akorn,AnaSed ®
  7. 肝素(Swiss Vault Engine,目录号:25021-400-66)
  8. 磷酸盐缓冲液(Sigma-Aldrich,目录号:P3619)
  9. Dulbecco的磷酸盐缓冲盐水(DPBS)(Sigma-Aldrich,目录号:D5652)
  10. 甘氨酸(分子量:75.07)(Sigma-Aldrich,目录号:410225)
  11. 正常驴血清(EMD Millipore,目录号:S30-100mL)
  12. Triton(Sigma-Aldrich,目录号:X100)
  13. 识别增强黄色荧光蛋白(EYFP)的绿色荧光蛋白(GFP)(EMD Millipore,目录号:AB3080)的兔多克隆抗体
  14. 识别多巴胺神经元特异性标志物的酪氨酸羟化酶(TH)(EMD Millipore,目录号:MAB318)的小鼠单克隆抗体
  15. 驴抗兔Alexa Fluor 488二抗(Thermo Fisher Scientific,Invitrogen,目录号:A-21206)
  16. 驴抗小鼠Alexa Fluor 594二抗(Thermo Fisher Scientific,Invitrogen,目录号:A-21203)
  17. ProLong Gold安装介质(Thermo Fisher Scientific,Molecular Probes TM ,目录号:P36930)
  18. 多聚甲醛(PFA,16%)(Electron Microscopy Sciences,目录号:15710)
  19. 甘油(分子量:92.09)(Sigma-Aldrich,目录号:G5516)
  20. 乙二醇(分子量:62.07)(Sigma-Aldrich,目录号:324558)
  21. Tris HCl(Sigma-Aldrich,目录号:T2413)

设备

  1. 刷(小油漆刷)
  2. 振动切片机(Leica Biosystems Nussloch,型号:VT1200S)
  3. 实验室振动筛(Benchmark Scientific,型号:BlotBoy)
  4. 幻灯片存储盒(Thomas Scientific,目录号:1202N78)
  5. 共焦扫描显微镜(Olympus,型号:Fluoview FV1000)

软件

  1. ImageJ( imagej.nih.gov/ij/download.html)(Abràmoff等人,2004)

程序

  1. 脑固定切片(1天)
    1. 注射3-5周后,用i.p.注射氯胺酮(100mg/kg) - 地拉嗪(15mg/kg)溶液
    2. 使用含有10,000IU肝素/L的1ml温磷酸盐缓冲液(30℃,0.1M,pH7.4),然后用5ml的磷酸盐缓冲液,然后用5ml的4%PFA在磷酸盐缓冲液中快速灌注。 br />
    3. 将大脑和后缀在4%PFA溶液中去掉一夜。
    4. 将多孔板的每个孔用1ml冷冻保护剂溶液填充
    5. 在50微米的振动切片机上切片大脑,以产生冠状切片
    6. 将部分收集到多孔板中。
    7. 将脑切片置于-20°C直至加工。

  2. 免疫荧光染色和成像(3天)
    1. 用PBS在多孔板中洗涤振荡器(12rpm)5分钟;重复两次。
    2. 将部分移动到具有甘氨酸(100mM; 1ml /孔)的新的多孔板中以淬灭醛,并将多孔板放在振荡器上30分钟。
    3. 在PBS中洗涤切片5分钟;重复两次。
    4. 将具有正常驴血清(10%)的块在具有0.1%Triton的PBS中在摇床中稀释2小时。每孔加入1ml这种封闭溶液。
    5. 在含有2%正常驴血清和0.02%Triton的PBS中稀释抗体,抗GFP(稀释度:1:2000)和抗TH(稀释度:1:5000)。
    6. 将切片移动到含有抗-GG和抗TH抗体(1ml /孔)的新的多孔板中,并将切片在4℃振荡器上孵育过夜(至少16小时;为了更好的结果,离开抗体孵化在周末)
    7. 在PBS中洗涤切片5分钟。重复两次。
    8. 在室温下,在振荡器上用抗兔和抗小鼠二次抗体(稀释度:1:200,PBS-T 0.02%)孵育2小时。
    9. 洗涤PBS中的切片5分钟;重复两次。
    10. 在磷酸盐缓冲液中载玻片上的片段,让它们干燥5分钟
    11. 当完全干燥时,将几滴安装介质放在部分和盖玻片上,小心不要产生气泡。
    12. 在黑暗中离开幻灯片,室温过夜。
    13. 在盖玻片的边缘周围涂抹指甲油;这是为了防止盖玻片移动,因为安装介质不会干燥。
    14. 在4°C的幻灯片存储箱中存放幻灯片。荧光信号通常持续2年。
    15. 使用共聚焦扫描显微镜对感兴趣的区域进行成像,在这种情况下,腹侧被盖区域和黑质符合密度(参见Mingote等人,2015年的图1)。使用鼠标图集来确保对所有受试者进行相同的区域成像。
    16. 采用共聚焦显微照片堆叠(60倍油物镜;光学变焦1.4倍和 - 步长增量0.42微米; 800×800像素像素大小为0.189微米的像帧 )通过整个组织部分(40至60张图像)。

数据分析

  1. 使用ImageJ线性调整共焦堆叠的对比度和亮度。
  2. 制作共焦堆叠的z投影图像(该图像将被用于计数),但是保持所有共焦图像堆叠的图像也会打开(此图像将用于识别不同类型的单元格)。
  3. 识别由几个共聚焦堆组成的每个图像中TH阳性(+)/ChR2-EYFP( - ),TH( - )/ChR2-EYFP(+)和TH(+)/ChR2-EYFP(+)细胞的数量向上和向下滚动z轴并比较红色和绿色通道中的图像。
  4. 使用ImageJ单元格计数器插件计算z投影图像中每种类型单元格的数量。
  5. 计算每个立体定位的功效和特异性百分比值。病毒转染的效力计算为TH(+)和ChR2-EYFP(+)相对于TH(+)细胞总数的数量。病毒转染的特异性计算为与ChR2(+)细胞总数相关的TH(+)和ChR2-EYFP(+)的数量(参见下面的图3,或者在Mingote等人的图2) al 。,2015)。
  6. 使用非参数弗里德曼测试分析沿前后侧和内侧 - 横轴的差异,其显着性设置为0.05。
  7. 识别感兴趣的细胞的其他部分的投影区域,在这种情况下是确定从...记录的区域的腹侧中脑多巴胺神经元。
     

    图3.确定ChR2表达在多巴胺神经元中的功效和特异性。
    来自侧面VTA和SNc的顶部面板,共焦z投影显微照片显示在洋红色中表达酪氨酸羟化酶(TH)的细胞,ChR2-绿色的EYFP和表达白色标记的细胞。在左侧的面板中,白色星星表示TH(+)和ChR2-EYFP(+);蓝色星星表示TH(+)和ChR2( - )细胞。左下方,腹侧中脑示意图显示相对于AAV注射部位拍摄显微照片的位置。基于在顶部的显微照片中完成的细胞计数的右下,功效和特异性图。位于离注射部位更远的多巴胺神经元的功效降低。使用AAV-ChR2-EYFP的特异性非常高; TH( - )和ChR2-EYFP-细胞的数量可以忽略不计(在这里看不到)。

笔记

  1. 据报道,IgG存在于小胶质细胞中,这导致用抗小鼠二抗的假标记;然而,也已经显示这种标记是小鼠应变特异性的,并且取决于所使用的免疫染色程序(Hazama等人,2005)。我们的免疫染色方案没有产生小胶质细胞的任何非特异性染色。或者,可以使用在鸡或兔中制备的抗TH抗体。
  2. 使用Ai32小鼠(B6; 129S-Gt(ROSA)26Sortm32(CAG-COP4 * H134R/EYFP)Hze/J; RRID:IMSR_JAX:012569)(Madisen等人),可以转基因表达ChR2, em>,,2012)。转基因策略从病毒注射得不到,从动物到动物产生可靠的表达(图4)。为了直接比较这两种靶向多巴胺神经元的方法,另见Tritsch等人。 (2012,补充图1)。然而,转基因策略中的ChR2特异性可能由于异位表达而受损。例如,使用血清素转运蛋白来驱动表达和ChR2转基因(,即SERT-cre :: Ai32小鼠)不是一个很好的策略来定位Raphe细胞核5-羟色胺神经元选择性地因为SERT启动子在发育期间在丘脑细胞中暂时活跃(Narboux-Nême等人,2008),并且这些细胞将在成年期继续表达ChR2。


    图4. ChR2-EYFP在DAT IREScre 小鼠腹侧中脑TH(+)神经元中的表达,通过双侧注射AAV-ChR2-EYFP(上)或使用Ai32小鼠(底部)进行育种。
    使用这两种策略,ChR2表达特异于TH(+)细胞。然而,病毒策略可能无法针对所有TH(+),如顶部面板显微照片的左侧所示。当使用病毒策略时,ChR2表达功效高度依赖于注射成功和每只注射的小鼠感兴趣的脑区域内病毒的传播。因此,转基因策略的表达效率和可靠性要高得多(下图)。刻度尺表示400μm。

食谱

  1. 4%多聚甲醛(PFA)溶液(20ml) 10ml 0.2M磷酸盐缓冲液
    5ml ddH 2 O
  2. 冷冻保护剂溶液(100ml)
    30毫升甘油
    30 ml乙二醇
    60 ml 0.1 M Tris HCl

第三部分:使用切片电生理学和光遗传学确定多巴胺神经元的兴奋性功能性连接体 >

材料和试剂

  1. Nalgene 4毫米注射器过滤器,0.2微米孔径,乙酸纤维素膜(Thermo Fisher Scientific,Thermo Scientific TM,目录号:171-0020)
  2. 1毫升结核菌素滑点注射器,1毫升(BD,目录号:309659)
  3. 用于填充微量移液器的非金属注射器针头,28 G,67 mm(世界精密仪器,目录号:MF28G67-5)
  4. 移液器提示
  5. 玻璃移液管
  6. 盐酸氯胺酮(Ketved,Vedco)
  7. 赛拉嗪(Akorn,AnaSed ®
  8. 氯化钠(NaCl)(分子量:58.44)(Sigma-Aldrich,目录号:746398)
  9. 氯化钾(KCl)(分子量:74.55)(Sigma-Aldrich,目录号:746436)
  10. 碳酸氢钠(NaHCO 3)(分子量:84.01)(Sigma-Aldrich,目录号:S6014)
  11. 磷酸二氢钠(NaH 2 PO 4)(分子量:119.98)(Sigma-Aldrich,目录号:S3139)
  12. 氯化钙脱水(CaCl 2)(分子量:147.01)(Sigma-Aldrich,目录号:223506)
  13. 氯化镁(MgCl 2)(分子量:95.21)(Sigma-Aldrich,目录号:M8266)
  14. 葡萄糖(分子量:180.16)(Sigma-Aldrich,目录号:G6152)
  15. Carbogen(95%O 2 + 5%CO 2)
  16. SR95531(加巴嗪,GABAA拮抗剂)(分子量:368.23)(Abcam,目录号:Asc-042)
  17. CGP55845(GABA 拮抗剂)(分子量:438.71)(Tocris Bioscience,目录号:1248)
  18. SCH23390(D1拮抗剂)(分子量:324.24)(Tocris Bioscience,目录号:0925)
  19. ( - ) - 舒必利(D2拮抗剂)(分子量:341.42)(Tocris Bioscience,目录号:0895)
  20. 东莨菪碱氢溴酸盐(毒蕈碱拮抗剂)(分子量:384.27)(Tocris Bioscience,目录号:1414)
  21. 利多卡因N,N-溴代(QX-314,细胞内钠通道阻断剂)(分子量:343.30)(Sigma-Aldrich,目录号:L5783)
  22. 6-氰基-7-硝基喹喔啉-2,3-二酮二钠盐(CNQX)(分子量:276.12)(Tocris Bioscience,目录号:1045)
  23. 葡萄糖酸(分子量:196.16)(Sigma-Aldrich,目录号:G1951)
  24. 氢氧化铯水合物(CsOH·H 2 O)(分子量:149.91)(Sigma-Aldrich,目录号:C8518)
  25. HEPES(分子量:238.30)(Sigma-Aldrich,目录号:RDD002)
  26. EGTA(分子量:380.35)(Sigma-Aldrich,目录号:E3889)
  27. ATP-Na 2(分子量:551.14)(Sigma-Aldrich,目录号:A2383)
  28. GTP-Na 2(分子量:523.18)(Sigma-Aldrich,目录号:G8877)
  29. 高糖aCSF(见配方)
  30. 标准aCSF(见配方)
  31. 细胞内溶液(见食谱)

设备

  1. 剪刀
  2. 振动切片机(Leica Biosystems Nussloch,型号:VT1200S)
    注意:不同的振膜用于组织学和切片记录,以避免甲醛对切片健康的不利影响。
  3. 移液器拉杆(Sutter Instruments,型号:P97)
  4. 贴片移液器,标准硼硅玻璃毛细管(OD/ID 1.5/0.84 mm),带灯丝(World Precision Instruments,目录号:1B150F-4)
  5. CCD相机(奥林巴斯,型号:OLY-150,停产),作为替代,具有实时成像模式的科学CMOS相机(例如,,Hamamatsu Photonics,型号:C11440-42U)可以使用
  6. 大功率蓝色LED(470nm; LED控制器电流设置为1A,对应于10V的LED电压)(Thorlabs,型号:DC4100)
  7. 荧光显微镜(Olympus,型号:BX61WI)具有60x浸水透镜
  8. 记录室(灌注室圆形)(Siskiyou,型号:PC-R)
  9. 参考电极(Ag-AgCl电极,1mm直径,3mm长)(World Precision Instruments,目录号:EP1)
  10. 微机械手(机械,赫ux黎式)(Siskiyou,型号:MX310R)
  11. 定制"竖琴"将切片固定到录音室(U形扁平铂 - 铱线与单个尼龙绳由氰基丙烯酸酯胶粘附)
  12. 浴液温度控制器(华纳仪器,型号:TC 344B)
  13. 贴片钳放大器(Molecular Devices,型号:Axopatch 200B)
  14. 计算机用于数据采集接口(HEKA Elektronik Dr. Schulze,型号:MICTECH ITC-18)或(Molecular Devices,型号:Axon DigiData 1500)

软件

  1. Axograph X(Axograph Scientific)或pClamp 10(Molecular Devices)

程序

  1. 切片录制准备(1天)
    1. 孵化脑切片时使用高葡萄糖人造脑脊髓液(aCSF)标准aCSF用于膜片钳记录。对于两者,准备10x的库存解决方案(参见食谱部分)。
    2. 用QX-314细胞内溶液制备葡萄糖酸铯(参见食谱部分)和等分试样。

  2. 制作脑切片和膜片钳记录(1天)
    1. 注射后3-5周,用氯胺酮(100mg/kg)+赛拉嗪(15mg/kg)麻醉小鼠。
    2. 用剪刀沿着矢状缝线打开颅骨,反射颅骨瓣,并将脑部移入冰冷的高葡萄糖aCSF(75mM NaCl,2.5mM KCl,26mM NaHCO 3,1.25mM NaH 2 PO 4,0.7mM CaCl 2,2mM MgCl 2和100mM葡萄糖;参见食谱部分),饱和碳水化合物
    3. 为每个感兴趣的区域制作300μm厚的切片。为了建立VTA多巴胺神经元的功能性连接物,在包含纹状体,扁桃体,海马结构或前皮层的四个不同冠状切片中进行记录。
    4. 在室温下用碳源饱和至少1小时的高葡萄糖aCSF预孵育切片进行恢复。
    5. 在孵育时间内,用移液管电阻拉伸移液器,介于4和8MΩ(尖端直径〜1μm)之间。
    6. 在荧光显微镜的阶段将一个切片转移到记录室(浸没,500μl体积),并用竖琴固定切片。应使用标准的aCSF(125mM NaCl,2.5mM KCl,25mM NaHCO 3,1.25mM NaH 2 PO 3)连续灌注记录室(1.5ml/min)亚> 4,2mM CaCl 2,1mM MgCl 2和25mM葡萄糖;参见食谱部分),并保持在31-33 °C。
    7. 谷氨酸能反应通过阻断多巴胺,GABA和乙酰胆碱的作用,加入到灌注液中的拮抗剂混合物分离:10μMSR95531,3μMCGP55345,10μMSCH23390,10μM( - ) - 舒必利和1μM东莨菪碱。
    8. 使用10x目标来关注感兴趣的区域,然后使用60x和相机来识别要修补的单元格。所有记录应在具有可见ChR2-EYFP轴突的区域进行。
    9. 使用钠通道阻滞剂(QX-314)在细胞内溶液中以-75 mV保持电位进行电压钳记录,以阻止有效电流。可以在细胞附着模式下监测细胞活性。一旦实现了全细胞模式,由于铯基移液管溶液(包括QX-314)阻止了内在活性,因此只能根据细胞体的位置,大小和形状鉴定神经元。
    10. 在定位健康的细胞后,使用非金属注射器针头用过滤的(0.2μm孔注射器过滤器)铯 - 葡萄糖酸铯细胞内溶液回填补片移液管,并将移液管安装在显微操纵器上的移液管支架中。将移液管吸头浸入浴液前,向玻璃移液管施加温和的正压;这可以防止由于溶液吸液和移液管堵塞而使细胞内溶液稀释
    11. 使用显微操纵器将移液器推入所选择的细胞。用移液管尖端接触细胞膜后,轻轻推进以使膜变暗。释放正压并确认尖端电阻增加,然后对移液管施加温和的负压,施加负电位以达到GΩ密封,等待1-2分钟使密封最大化。施加进一步的负压使得移液管末端的膜破裂以进行全细胞记录,在穿透后迅速释放移液管的负压。
    12. 为了最小化基线噪声以更好地检测小振幅兴奋性突触后电流(EPSCs),串联电阻不被补偿(见注释)。
    13. 使用Axograph X或pClamp的数据采集在达到全细胞模式后5分钟开始,以允许细胞内溶液扩散到记录的细胞中。
    14. 以每10秒(0.1Hz)的大功率蓝色LED照明的短暂闪烁(0.1Hz的5毫秒)进行光刺激以引起突触反应。为了监控串联电阻,在每次光刺激前50毫秒,施加5 mV,5毫秒的电压脉冲。每个单元记录至少20个响应。
    15. 戊巴比妥反应应通过灌注40μMCNQX进行确证,药物应用后至少5分钟即可全面应用。

数据分析

  1. 对于每个细胞,平均10个连续痕迹。丢弃第一条迹线,因为由于突触连接的休息状态,EPSC幅度总是显着更大。
  2. 测量50毫秒后光刺激时间窗口内的峰值幅度。
  3. 用于确定连接的检测阈值应设置为在光刺激之前的100毫秒时间窗口中的基线(包括自发的EPSCs)振幅的平均值±2个标准偏差。当后刺激EPSCs的峰值振幅超过该阈值时,细胞计数为具有多巴胺神经元谷氨酸能连接(见图5)。


    图5.确定多巴胺神经元谷氨酸能连接的强度和发生率。 A-C。痕迹显示无连接记录的细胞(锥体细胞1)和具有不同强度连接的两个细胞(锥体细胞2和3;蓝色痕迹)的实例。 AMPA /红藻氨酸受体拮抗剂CNQX(40μM)完全阻断显示谷氨酸调节的光刺激EPSC(锥体细胞3;红色痕迹)。灰色痕迹用于计算基线的平均幅度加上2个标准偏差(SD);超过此阈值的响应被认为显示连接。 D.右下图,是该图所示数据的图。连接的强度被计算为具有连接的两个小区中的响应的平均幅度,而连接的发生率是显示与所记录的所有细胞的连接的小区的百分比,在这种情况下为2的3细胞。记录显示来自内嗅皮层。光刺激由蓝色条表示。

笔记

  1. 洗浴液和移液管可产生约15 mV的液体电位。要在线校正结电位并确定-75 mV的保持,将放大器的保持命令设置为-60 mV(Neher,1992)。
  2. 由于串联电阻补偿使用正反馈电路,在线补偿会增加基线噪声。一般来说,如果响应小,慢,则串联电阻补偿不会影响响应的幅度或时间过程,这是我们确认的。然而,串联电阻补偿应考虑在线或离线记录更大更快的响应(使用分析软件)。
  3. 为了改善突触后细胞的鉴定,可以用GFP遗传靶向记录的细胞,或者用可固定的标记(例如,biocytin,Alexa荧光染料)填充记录细胞,然后固定用已知细胞标记物双重标记细胞的组织。

食谱

  1. 10x aCSF无葡萄糖,镁或钙的储备溶液
    43.83克NaCl
    1.86克KCl
    21.84g NaHCO 3
    1.5g NaH 2 PO 4
    加入ddH 2 O以制成1升溶液并将其储存在4°C
  2. 高糖aCSF(1升)
    在切片记录实验的当天使高糖aCSF
    稀释10倍储备溶液10次(100ml 900ml ddH 2 O中的储备溶液)
    加入18.02 g葡萄糖 加入2ml 1M MgCl 2水溶液(加入95.21mg/1ml ddH 2 O)
    加入0.7ml 1M CaCl 2溶液(加入147mg/1ml ddH 2 O)
  3. 标准aCSF(1 L)
    在切片记录实验的当天使标准aCSF
    稀释储备溶液10次(100ml储存液在900ml ddH 2 O中)
    加入4.5 g葡萄糖 加入1ml 1M MgCl 2溶液(加入95.21mg/1ml ddH 2 O)
    加入2ml 1M CaCl 2溶液(加入147mg/1ml ddH 2 O)
    如果加入CaCl 2,会导致碳酸钙沉淀,使碳水溶液饱和以清除沉淀物。
  4. 细胞内溶液(含有QX-314的葡萄糖酸铯,25ml),渗透压287mOsm
    1ml 3.25M葡萄糖酸(Sigma-Aldrich溶液的摩尔浓度)
    3.25ml 1M CsOH溶液(每1ml ddH 2 O加入130mg)
    加入50μl1M MgCl 2溶液(加入95.21mg/1ml ddH 2 O)
    2.5μl1M CaCl 2溶液(每1ml ddH 2 O加入147mg)
    2.5ml 50mM QX-314溶液(加入17.2mg/1ml ddH 2 O)
    250μl1M HEPES溶液(加入238.30mg/1ml ddH 2 O)
    9.5毫克EGTA
    加入17.948毫升ddH 2 O -/- 让溶液在4℃下静置过夜以稳定pH值
    使用CsOH溶液将pH调节至7.3 加入25.4mg的ATP-Na 2
    加入3.9mg GTP-Na 2
    用2μm过滤器过滤溶液
    制成1毫升等分试样,并将其储存在-20°C

致谢

该方案用于获得"神经科学杂志"(Mingote,S.,Chuhma,N.,Kusnoor,S.V.,Field,B.,Deutch,A.Y.and Rayport,S。[2015])中公布的数据。前脑区多巴胺神经元谷氨酸能连接的功能连接分析。 J Neurosci 35 [49]:16259-16271)。根据哥伦比亚大学和纽约州精神病学研究所机构动物护理和使用委员会批准的方案,按照国家实验动物护理和使用指南健康指南的指导方针进行小鼠手术。这项工作得到了NARSAD青年研究者奖(SM),DA017978和MH087758(SR)的支持。

参考文献

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  5. Faget,L.,Osakada,F.,Duan,J.,Ressler,R.,Johnson,AB,Proudfoot,JA,Yoo,JH,Callaway,EM和Hnasko,TS(2016)。< a class = ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/27292633"target ="_ blank">传入输入到腹侧被盖区域的神经递质定义的细胞类型。 em> Cell Rep 15(12):2796-2808。
  6. Hazama,GI,Yasuhara,O.,Morita,H.,Aimi,Y.,Tooyama,I。和Kimura,H。(2005)。小鼠脑IgG样免疫反应性:小胶质细胞中的菌株特异性发生和IgG的生物化学鉴定。 Comp Neurol 492(2):234-249。
  7. Hnasko,TS,Hjelmstad,GO,Fields,HL和Edwards,RH(2012)。  腹侧覆盖区谷氨酸神经元:电生理特性和预测。 Neurosci 32(43):15076-15085。
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  9. Mingote,S.,Chuhma,N.,Kusnoor,SV,Field,B.,Deutch,AY and Rayport,S。(2015)。< a class ="ke-insertfile"href ="https: .ncbi.nlm.nih.gov/pubmed/26658874"target ="_ blank">前脑区域多巴胺神经元谷氨酸能连接的功能连接分析。 Neurosci 35(49):16259 -16271。
  10. Narboux-Nême,N.,Pavone,LM,Avallone,L.,Zhuang,X。和Gaspar,P。(2008)。液体接头修正膜片钳实验的潜力。 方法Enzymol 207:123-131。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Mingote, S., Chuhma, N. and Rayport, S. (2017). Optogenetic Mapping of Synaptic Connections in Mouse Brain Slices to Define the Functional Connectome of Identified Neuronal Populations. Bio-protocol 7(1): e2090. DOI: 10.21769/BioProtoc.2090.
  2. Mingote, S., Chuhma, N., Kusnoor, S. V., Field, B., Deutch, A. Y. and Rayport, S. (2015). Functional connectome analysis of dopamine neuron glutamatergic connections in forebrain regions. J Neurosci 35(49): 16259-16271.
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