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Aug 2020
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Electrophysiological Properties of Neurons: Current-Clamp Recordings in Mouse Brain Slices and Firing-Pattern Analysis
神经元的电生理特性:小鼠脑切片中的电流钳记录和放电模式分析   

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Abstract

Characterization of an electrically active cell, such as a neuron, demands measurement of its electrical properties. Due to differences in gene activation, location, innervation patterns, and functions, the millions of neurons in the mammalian brain are tremendously diverse in their membrane characteristics and abilities to generate action potentials. These features can be measured with a patch-clamp technique in whole-cell current-clamp configuration followed by detailed post-hoc analysis of firing patterns. This analysis can be time-consuming, and different laboratories have their own methods to perform it, either manually or with custom-written scripts. Here, we describe in detail a protocol for firing-pattern registration in neurons of the ventral tegmental area (VTA) as an example and introduce a software for its fast and convenient analysis. With the help of this article, other research groups can easily apply this method and generate unified types of data that are comparable between brain regions and various studies.


Graphic abstract:



Workflow of the Protocol


Keywords: Patch-clamp (膜片箝), Current-clamp (电流钳), Action potential (动作电位), Single-cell electrophysiology (单细胞电生理学), Acute brain slices (急性脑片), Ventral tegmental area (腹侧被盖区), Automated firing pattern analysis (自动放电模式分析)

Background

The main feature of a neuron is its ability to engage in fast chemo-electrical communication with other cells. The unique neuronal membrane constitution with a high density of ion channels and other specific proteins allows the generation of an action potential within milliseconds after receiving an adequate input. Therefore, characterization of any neuron would be incomplete without description of the electrical properties of its membrane. For this purpose, we can use a convenient “patch-clamp” technique (Neher and Sakmann, 1976), which resulted from centuries of evolution in electrophysiological methods (Verkhratsky and Parpura, 2014). A big advantage of this method is the possibility to combine it with other modern single-cell approaches and collect all necessary data for defining a neuron’s type. Filling of the cell with intracellular dyes through the patch pipette (Marx et al., 2012) and collecting the cell’s aspirate after electrophysiological registration (Sucher and Deitcher, 1995; Fuzik et al., 2016) allow the simultaneous reconstruction of 3D morphology and analysis of mRNA content and firing pattern of the same cell. There are several types of patch-clamp configurations, but we focus here on the whole-cell current-clamp modification, which allows registering changes in the membrane voltage while controlling the electrical current flow; in other words, it allows registering action potentials (AP) in response to specific current injections.


The whole-cell current-clamp configuration of the patch-clamp technique is a well-established method that has been used for decades in the characterization of intrinsic membrane properties of electrically active cells (Neher and Sakmann, 1976; Andrew, 1986; Sanchez-Aguilera et al., 2020). Although most of the researchers are interested in a similar set of parameters and use similar protocols, the experimental details and final data are variable and, therefore, hard to compare (see https://neuroelectro.org/article/index). While preparing to describe somatostatin-expressing neurons in the mouse ventral tegmental area (VTA) for our recent article (Nagaeva et al., 2020), we tried to collect a maximal set of membrane properties for firing pattern analysis based on previous publications (Halabisky, 2006; Ma et al., 2006; Wierenga et al., 2010). The list of these properties can be found in Nagaeva et al., 2020, Appendix Table 1. Similarly, we made a current-stimulation protocol that allowed registering all these properties in one short run. Additionally, we developed a MatLab plugin for fast and convenient extraction of all these parameters.


Our protocol article aims to provide a clear workflow for firing pattern analysis and includes all steps from the preparation of acute brain slices to the final data extraction. It can be applied for electrophysiological studies of previously unknown neurons or as part of the currently popular patch-seq approach (Cadwell et al., 2016; Gouwens et al., 2020). For newly described neurons, the extracted data can be further used for neuron subtyping according to their electrical membrane properties. To do so, one just needs to upload the resulting tables to a clustering algorithm previously published by our group (see further Nagaeva et al., 2020; https://github.com/elifesciences-publications/clustering-for-nagaeva-et.-al.-sst-vta). This protocol will simplify the firing pattern registration and analysis in future studies.


Materials and Reagents

  1. Preparation of acute brain slices

    1. Beakers: 1 L, 2 × 250 ml (VWR, catalog numbers: 213-1128, 213-1124)

    2. Petri dish 60 × 18 mm (VWR, catalog number: 734-2815p)

    3. Tubes for carbogen delivery into solution (Ismatec, catalog number: MF0028)

    4. Paint brush (VWR, catalog number: 470020-430)

    5. Pasteur pipette with a wide neck (you can simply cut off the tip) (Sarstedt, catalog number: NC9891525)

    6. Super glue (Loctite, catalog number: 230992)

    7. Filter paper (Whatman, WHA1001110)

    8. A box full of crushed ice

    9. Medical spatula with smooth ends 150 × 40 × 6 mm (Bochem, VWR catalog number: 231-0601)

    10. Carbogen (95% O2 + 5% CO2)

    11. Floating net for brain slices

      A nice one can be 3D-printed from here https://3dprint.nih.gov/discover/3dpx-001623.

    12. Reagents for Artificial Cerebrospinal Fluid (ACSF) and Cutting solution:

      1. NaCl (Fisher BioReagents, catalog number: BP358)

      2. KCl (Amresco, catalog number: 0395)

      3. MgCl2·6H2O (Fisher BioReagents, catalog number: BP214)

      4. NaH2PO4·H2O (Merck, catalog number: 1.0634)

      5. NaHCO3 (Sigma-Aldrich, catalog number: 31437)

      6. D-(+)-Glucose (Alfa Aesar, catalog number: A16828)

      7. Sucrose (Fisher Scientific, catalog number: 10634932)

      8. CaCl2 (Amresco, catalog number: 0556)


  2. Electrophysiology

    1. Beakers: 250 and 25 ml (VWR, catalog numbers: 213-1128, 213-1120)

    2. 1-ml micropipette (Thermo Scientific, Finnpipette F2)

    3. 1.5-ml Eppendorf tubes for intracellular solution (Eppendorf, catalog number: 0030120086)

    4. Glass capillaries with filament (World Precision Instruments, catalog number: TW150F-4)

    5. Paint brush (VWR, catalog number: 470020-430)

    6. Silver/platinum wire or special net for slice holding in the microscopy chamber (see Figure 9)

    7. 1 ml syringe (Terumo, catalog number: SS+01T1)

    8. Syringe PVDF Durapore filter (Merck Millipore, catalog number: SLGV013SL)

    9. Plastic tubing ID 010 × OD 030 (Tygon, catalog number: AAD04091)

    10. Reagents for Intracellular solution (IS):

      1. K-gluconate (Sigma-Aldrich, catalog number: P1847)

      2. HEPES (Alfa Aesar, catalog number: A14777)

      3. EGTA (Sigma-Aldrich, catalog number: E4378)

      4. Na2-ATP (Sigma-Aldrich, catalog number: A6419)

      5. Na-GTP (Sigma-Aldrich, catalog number: G8877)

      6. Na2-phosphocreatine (Sigma-Aldrich, catalog number: P7936)

      7. KOH (Sigma-Aldrich, catalog number: 221473)

    11. ASCF solution (see Recipes)

    12. Cutting solution (see Recipes)

    13. Intracellular solution (see Recipes)

Equipment

  1. Volumetric flask 1 L (Brand, VWR catalog number: 612-5082)

  2. Big scissors (Fiskars, catalog number:1005151)

  3. Small scissors (Bochem, VWR catalog number: 233-2121)

  4. Small tweezers 105 mm (Usbeck, VWR catalog number: 232-0094)

  5. Scalpel (Swann-morton, VWR catalog number: swan0565)

  6. Teaspoon

  7. Razor blade for the vibratome (World Precision Instruments, catalog number: 752-1-SS)

    The same can be used for the brain dissection in Step A2b of the Procedure section.

  8. Laboratory scale (0.001-100 g; Mettler PJ360 DeltaRange)

  9. pH-meter (Metrohm, 827 pHlab)

  10. Magnetic stirrer (Merck, IKA big-squid)

  11. Laboratory water bath (Grant Instruments, Bath JB Aqua 12 Plus)

  12. Vibratome (Thermo Scientific, Microm HM650V)

  13. Osmometer (Advanced Instruments Inc., Model 3320)

  14. Micropipette puller (Sutter Instruments, Model P-1000)

  15. Epifluorescent microscope (Olympus, BX51WI)

  16. Fluorescent light source – 100 W mercury arc lamp with a power supply (Olympus, U-RFL-T)

  17. CCD camera (Sony, XC-E150)

  18. Heat controller (Warner Electric, TC-324B single channel)

  19. Laboratory vacuum pump (KNF, N 811 KTP)

  20. Amplifier (Molecular Devices, Axon Instruments, Multiclamp 700B)

  21. Digidata (Molecular Devices, Axon Instruments, Model 1322a)

  22. Micromanipulators (Sensapex, µMp-3)

Software

  1. pClamp 8.2 (or later) package (Molecular Devices, https://www.moleculardevices.com/products/axon-patch-clamp-system/acquisition-and-analysis-software/pclamp-software-suite#Resources)

  2. Software for your microscopy camera

  3. MATLAB R2018b (Mathworks, https://se.mathworks.com/products/matlab.html)

  4. Microsoft Excel 2016 (Microsoft, https://www.microsoft.com/en-ww/microsoft-365/excel)

Procedure

  1. Preparation of acute brain slices

    Note: Here, we prepare horizontal slices of the mouse midbrain, aiming to patch dtTomato-positive fluorescent cells from the VTA. The reader can prepare slices from any other brain area using the same reagents and procedure.

    1. Prepare Artificial Cerebrospinal Fluid (ACSF) and Cutting solutions (see Recipes) if you use juvenile mice younger than P30. Store solutions not longer than 3-4 days at 4°C.

      Note: If you are using older animals, see Ting et al. (2018).

      1. Put all powders except CaCl2 in 1-L beaker and mix with 800 ml MilliQ water using a magnetic stirrer. Add CaCl2 after all other powders are fully dissolved.

      2. Pour the solution into 1-L volumetric flask and adjust the volume. Adjust pH to 7.3-7.4 by bubbling with carbogen.

    2. Prepare your working space for brain dissection.

      1. Place “dry” instruments in a convenient order to reach them fast: big scissors, small scissors, small tweezers, scalpel, bent piece of filter paper, and waterproof superglue (Figure 1).



        Figure 1. Set of “dry” instruments


      2. Put the instruments – teaspoon, razor blade, medical spatula with narrow, smooth ends, and 60-mm Petri dish covered by filter paper – and approximately 250 ml of cutting solution on ice (Figure 2).

        Note: The ACSF solution should be chilled to 0°C beforehand and have floating pieces of ice. Aerate the solution with carbogen for 5 min before and during surgery (Figure 2).

        Tip: Sharp and convenient instruments ensure the success of any surgical operation. All instruments should be cleaned with MilliQ water after use.



        Figure 2. Set of “cold” instruments


      3. Put a 250-ml beaker with constantly aerated 200 ml of ACSF solution and a floating net in a water bath at 33°C (Figure 3).



        Figure 3. Beaker filled with ACSF solution and a floating net for incubation of brain slices at 33°C


      4. Prepare vibratome: insert the blade and tune the program. It is convenient to have a small paint brush, small tweezers, and a Pasteur pipette with a wide neck near the vibratome (Figure 4).

        Note: We have used a vibratome program with the following parameters: feed=225 µm, frequency=88 Hz, amplitude=0.9 mm, and velocity=0.9 mm/s.



      Figure 4. Set of instruments for brain slicing


      We recommend having all necessary equipment and instruments at hand range as you would need to move between them quickly. When everything is ready, it is time to start the brain dissection.


    3. Brain dissection (approx. 1 min).

      Notes:

      1. As the brain cells are very sensitive to hypoxia, and the metabolism is faster in warm environment, it is essential to transfer the brain from a live animal to cold cutting solution as rapidly as possible. We recommend using a stopwatch in the beginning, aiming to finish the whole procedure within 60-80 s.

      2. Important note: Consult your local animal welfare authorities for information on the appropriate anesthesia type to use before animal decapitation.

      3. For brain research purposes, authorities sometimes allow fast physical euthanasia of neonatal/juvenile mice with the method of decapitation, but it should be only performed by well-trained personnel.


      1. Decapitate the mouse with big scissors in one move.

      2. Cut the skin from the neck to between the eyes with small scissors.

      3. Cut the skull from the back to the bregma along the midline with the same small scissors. Be careful not to touch the brain surface with the scissors.

      4. Open the skull half by half with small tweezers from the midline to the side.

        Note: It is very important to choose appropriate tweezers and use them carefully, especially if your target is the cerebral cortex.

      5. Use the scalpel to cut out unnecessary parts of the brain within the skull. We cut out half of the cerebellum and frontal pole, as shown in Figure 5.

        Note: From here on, all cutting procedures aim at getting midbrain horizontal slices and should be revised for obtaining other brain regions of interest.



        Figure 5. Schematic representation of mouse head with the brain inside. Dashed lines illustrate scalpel cuts.


        Note: Here, we switch to “cold” instruments and environment to slow down the metabolism.

      6. Take out the brain from the skull with the help of a medical spatula and immediately transfer it into the cold aerated cutting solution. Leave it there for 1 min while preparing the cold vibratome platform for supergluing the brain.

    4. Brain slicing (max 5-10 min).

      Note: Background knowledge on the use of the vibratome is required to perform brain slicing.

      1. Take out the brain from the cutting solution with a teaspoon and put it onto the Petri dish with the cortex facing down (that is, “on the cortex”). Then, you can cut out all unnecessary parts with a cold razor blade if you did not do so already before taking the brain out from the skull (see above).

      2. Put a small drop of superglue onto the vibratome platform and immediately transfer the brain onto it with the help of the bent piece of filter paper. Do not change brain orientation (ventral part is up) and glue it so that the cerebellum is facing the vibratome blade.

      3. Place the platform into the buffer tray and fill up the tray with the cold aerated cutting solution (Step A3f).

      4. With the help of small tweezers, carefully take off the filter paper, which still is stuck to the brain.

        Note: If you would like to extend the time of slicing, you can aerate the cutting solution also in the buffer tray.

      5. When the brain is placed as shown in Figure 6, it is ready for making horizontal midbrain slices. Carefully cut away unnecessary upper slices with 225-µm steps until the brain and cerebellum are fully connected. At this level [corresponds to -4.72 from Bregma (Franklin and Paxinos, 2008)], you will also see a distinctive “circle” in the center of the slice (see Figure 7). The next slice is the first midbrain slice that contains the VTA.



        Figure 6. Orientation of the brain inside the vibratome tray. The ventral part up and cerebellum is facing the blade. The right part of Figure 6 shows a close-up of the brain inside the tray.



        Figure 7. The correct level to start cutting horizontal VTA-containing slices (approximately at -4.72 from Bregma)


      6. It is possible to get two or three 225-µm-thick horizontal slices from the mouse VTA, which correspond to Bregma levels of about -4.72 mm, -4.56 mm, and -4.28 mm.

      7. Transfer the resulting slices into 33°C ACSF solution with the help of a Pasteur-pipette immediately after cutting each slice.

      8. Incubate the slices at 33°C for 60 min; then, keep the beaker with slices at room temperature. The slices are viable for at least 4 h. We start electrophysiology right after 60 min of incubation at 33°C.

        Note: It is critical that during incubation the slices are continuously aerated but do not float around because of the bubbles.


  2. Electrophysiology

    Note: Previous theoretical knowledge on electrophysiology and technical training in cell patching are required to perform further protocol steps.

    1. Prepare K-gluconate-based Intracellular solution (IS).

      1. Mix all reagents with 15 ml of MilliQ water in a 20-ml beaker.

      2. Put the beaker on ice before adding ATP, GTP, and phosphocreatine.

        Note: From here on, try to keep the solutions cold all the time, as ATP and GTP are sensitive to temperature.

      3. While mixing with a magnetic stirrer, measure the pH and adjust to 7.2 with KOH.

      4. Measure osmolarity and adjust it to 285 mOsm by adding MilliQ water milliliter by milliliter.

      5. Aliquot the IS solution in 1.5-ml Eppendorfs and store at -20°C for a maximum 2-3 months.

        Note: Thaw an Eppendorf tube containing IS every time just prior to the experiment and remember to keep it cold during the experiment (on ice or in the fridge at 4°C).

    2. Prepare 3-5 MΩ glass electrodes from borosilicate capillaries according to puller manufacturer’s instructions. We recommend preparing electrodes with a 4-step program. Figure 8 depicts our program for the Sutter P-1000 puller as an example. Note that parameters might be different depending on the capillaries’ RAMP test result (melting point temperature) and puller type.



      Figure 8. Example of the Sutter P-1000 puller program for suitable glass electrode preparation. Please note the RAMP.


    3. Prepare your electrophysiological setup for patching.

      1. Switch on the computer, fluorescent lamp, bright-field lamp, camera, solution heat controller, vacuum pump, amplifier, digidata, and manipulators.

      2. Prepare a 250-ml beaker with a constantly aerated ACSF solution. Tune the perfusion speed to 1-3 ml/min for a 1-ml recording chamber. Tune the heat controller to keep the solution at 33°C.

    4. Patching.

      1. Transfer a slice from the incubation beaker into the recording chamber with a wide-neck Pasteur pipette.

      2. Place the brain slice in the middle of the chamber with the cerebellum in its lower part, as shown in Figure 9. Press the slice down with a silver wire or special net slice holder.

        Note: The brain slice should be at the bottom of the recording chamber and remain stationary during the solution flow.



        Figure 9. Orientation of the brain slice within the recording chamber with the cerebellum toward the researcher. A silver wire holds the slice at the bottom of the chamber.


      3. Fill up the glass electrode with cold IS solution and attach it to the headstage.



        Figure 10. Example of the custom-made syringe for filling the intracellular solution


        Tip: You can make a very convenient syringe for IS filling (Figure 10) by using a 1 ml syringe, a Merck Millex Durapore filter, and a piece of flexible plastic tubing ID 010 × OD 030.


      4. Make positive pressure in the patch pipette by blowing into the tube and close it with a valve (Figure 11).

        Tip: To check whether the pressure is good enough, you can open the valve near your ear. If the pressure is good, you should hear a hollow flop.



        Figure 11. The tube and valve set is connected to the headstage for making positive/negative pressure inside the glass electrode


      5. Put the electrode down into the solution and check the resistance of the pipette in the Seal Test window (Figure 12). It should be 3-5 MΩ. Reset the pipette offset to 0 mV.

        Note: From here to step (v), the amplifier is in Voltage Clamp (VC) mode. While searching for a cell, the voltage is not clamped.



        Figure 12. Seal test window showing the pipette resistance of 4.3 MΩ


      6. Locate the pipette over the region of interest under small magnification, e.g., 5×. To patch cells in the VTA, locate the pipette over the region shown in Figure 13.



        Figure 13. VTA region (marked with green dashed line on the left) under a microscope 5× magnification
        Note: For convenience, the right image shows the same midbrain region marked with a dark dashed line within the horizontal brain slice at -4.72 cm from the bregma (Franklin and Paxinos, 2008).


      7. Go down with the pipette at 4/5 manipulator speed until the pipette is slightly above the slice focus.

      8. Switch to a higher magnification (e.g., 40×) and find the pipette.

      9. Switch to camera view.

      10. Slow down manipulator speed to 2-3/5 and slowly go down towards the slice surface, first with the objective and then with the pipette.

      11. Stop the pipette right above the slice surface.

        Tip: At this level, you can already evaluate the condition of the slice. If there are many bubbled cells on the surface, it might be difficult to find a healthy cell in the slice.

      12. Dim the light and switch to fluorescence. Search a healthy neuron with the objective. Do not move the objective too far; otherwise, it will be difficult to find your pipette again.

        Note: An unhealthy neuron is usually round, has clear visible nuclei, and an unclear or, sometimes, very contrasting contour.

      13. After finding a suitable neuron, remember its location. You can put some sign on the screen or mark it otherwise (Figure 14).



        Figure 14. Neurons expressing a fluorescent marker protein under the microscope at 40× magnification. The blue cross indicates a neuron suitable for patching.


      14. Go up with the objective and find the pipette. Move it to the neuron’s location above the slice.

      15. Blow into the tube to make sure there is a positive pressure inside the pipette. Reset the pipette offset to 0 mV once more. Pipette resistance must be stable all the time you go down towards the neuron.

      16. Go down to the cell at the slowest manipulator speed. Follow the pipette resistance.

      17. When the resistance rises by 0.1-0.2 MΩ (you might also see a small black dot on the cell, which corresponds to the pipette shadow), release the pipette pressure by opening the valve. Simultaneously, make a slight negative pressure in the pipette by sucking or with the help of a syringe.

      18. At this point, resistance rises to GΩ values (named gigaseal) (Figure 15), which is the first indication of a successful patch.



        Figure 15. Seal test window showing the gigaseal configuration (Seal Resistance = 2.7 GΩ)


      19. Clamp the holding potential at -70 mV; compensate fast and slow capacitances by clicking c-fast and c-slow buttons to get rid of transient currents. Hold the cell at gigaseal for 1-2 min to make the contact more stable.

      20. Break the membrane by making a strong but short suction to establish a whole-cell configuration.

        Note: At this point, the electrode and neuron become one electrical unit. This allows registration of the currents flowing through the neuronal channels and/or changes in the whole-cell membrane voltage.

      21. Immediately after the breakage, switch to the Membrane Test window and read the cell parameters (Figure 16).

        Note: It is good to know the cell’s capacitance (Cmin pF), access resistance (Rain MΩ), and holding current (Holdin pA).

        1. Cmmay vary depending on the cell size and usually is the 10-100 pF range. You can use this parameter for further characterization of the neuron along with other parameters extracted from the firing pattern analysis.

        2. Rashows the quality of the contact between the glass electrode and cell membrane; it should not exceed 20 MΩ. If it does, just try to suck a bit one more time and re-brake the membrane.

        3. Holdshows the applied current that is required to hold the cell at a certain voltage (-70 mV in our case); this value should be between 0 and -50 pA.

      22. Switch to the I=0 mode and read the neuron’s Resting Membrane Potential (RMP) in the MultiClamp software window V (mV).

        Note: Please, make a note of the liquid junction potential (LJP), which appears between two liquids with different ionic compositions. For extracellular ACSF and IS solutions used here, LJP is +12 mV. Usually, we do not correct it during recordings but always mention it in the Method section.



        Figure 16. Membrane Test window showing a successful whole-cell configuration in voltage-clamp mode and several cell parameters


      23. Switch to the Current Clamp (IC) mode and run the current-steps protocol provided below (Figure 17).

        Notes:

        1. We do not recommend injecting any current in the current-clamp mode unless you are sure of the normal RMP for a certain cell type.

        2. The episodic stimulation protocol lasts 2 s. It includes an 800-ms current step ranging from -60 to 680 pA in 10-pA increments and a 1.2-s recovery pause to allow the cell to get back to its normal RMP. This pause might be longer, depending on cell type and stimulation intensity.

        3. If you want to define AP threshold and rheobase current more precisely, you can additionally run a similar protocol from 0 to 30-50 pA in 1-pA increments.



      Figure 17. Recommended protocol for current-steps: 800-ms current steps from -60 pA to +680 pA in 10-pA increments

Data analysis

The features from raw *.abf traces are extracted in a semi-automatic way using FFFPA (fast-forward firing pattern analysis), an open-source plugin for MATLAB. FFFPA provides a flexible method to detect the most common parameters of APs (including peaks, thresholds, and AHPs) (Figure 18) and compute the most frequently analyzed features automatically, allowing to process of large batches of files quickly and accurately. Results of the automatic detection are visualized and can be inspected and adjusted manually if needed. Detailed definitions of the extracted features are summarized in Nagaeva et al., 2020, Appendix Table 1.



Figure 18. Basic parameters of action potential (AP), which are detected automatically by the FFFPA plugin. For definition of the parameters, see Nagaeva et al., 2020, Appendix Table 1.


Installation

  1. Installation requires MATLAB 2018a or a newer version with Signal Processing and Curve Fitting toolboxes.

  2. Download the FFFPA plugin from https://github.com/zubara/fffpa and unzip it to a local directory.

  3. Double-click on the ‘fffpa.mlappinstall’ file from MATLAB file explorer.

  4. Click “Install” in the opened dialog window.

  5. Once installed, open the app from the MATLAB Apps toolstrip. The graphical user interface for data import will appear (Figure 19).



    Figure 19. FFFPA graphical user interface for data import


Data import

  1. Click “Select files” and choose one or several ‘.abf’ files to process.

  2. Specify the sampling frequency in Hz and the time of the onset and the offset of the injected current step in seconds.

  3. Choose the sweeps that you wish to analyze. FFFPA can detect, for example, the rheobase current step defined as the first sweep where APs occurred, or the saturation current step defined as the sweep with the maximum number of APs.

  4. Specify the summary statistics for the analyzed APs. If multiple sweeps are analyzed, these options can differ between the first and other sweeps.

  5. Specify the filename and the desired output format.


Event detection and feature extraction
  1. (Optional) Go to the ‘Advanced’ tab (Figure 20) to specify the features that you wish to extract from the traces. Detailed definitions of the extracted features are summarized in Nagaeva et al., 2020, Appendix Table 1.



    Figure 20. FFFPA feature selection and advanced options tab


  2. Click the ‘Run’ button on the ‘Main’ tab (Figure 19).


Inspection and manual correction of detection results
  1. To inspect the results of automatic feature detection, go to the ‘Data viewer’ tab (Figure 21). This tab opens automatically after clicking ‘Run’ once the data is processed.



    Figure 21. FFFPA Data viewer tab


  2. The plot area displays the raw trace and the detected events AP peaks (blue circles), activation thresholds (red), afterhyperpolarization (AHP, cyan), and afterdepolarization (ADP, not shown). Please note that, by default, the ADP is only detected after the first AP or train of APs at rheobase.

  3. Use the ‘Next’ button to go through all sweeps of all analyzed cells consecutively or use the ‘cell ID’ and ‘sweep #’ fields to navigate to the specific sweep of the specific cell manually.

  4. If needed, the Data viewer allows you to correct the detection results. To do this, first click ‘Edit,’ then click the element that you want to adjust and specify its new position by clicking on the trace. To correct another point, click “Z” to activate the editing option again. After all corrections have been made, press ‘X’ to save the results.

  5. You can also discard the current cell from the output by unchecking the ‘Save to output’ box.

  6. Once satisfied with the results, click ‘Save selected.’ This step will compute features based on the (adjusted) detections and save the output file to disk. The summary file can be found in the same directory as the input data.

Notes

You can watch a short tutorial for the FFFPA plugin here: https://vimeo.com/497798349.

Recipes

Note: All recipes should be made in MilliQ water.

  1. Artificial Cerebral Spinal Fluid (ACSF) solution (Table 1)


    Table 1. Artificial Cerebral Spinal Fluid (ACSF) solution recipe

    ASCF solution Concentration in mM g/L
    NaCl 126 7.36
    KCl 1.6 0.12
    MgCl2·6H2O 1.2 0.243
    NaH2PO4·H2O 1.2 0.166
    NaHCO3 18 1.5
    D-Glucose 11 1.98
    CaCl2 2.5 0.37
    Carbogen needed for pH adjustment

    final pH 7.3-7.4

    Final osmolarity:
    300-310 mOsm


  2. Sucrose-based cutting solution (Table 2)


    Table 2. Sucrose-based cutting solution recipe

    Cutting solution Concentration in mM g/L
    NaCl 60 3.506
    KCl 2 0.148
    MgCl2·6H2O 8 1.626
    NaH2PO4·H2O 1.2 0.172
    NaHCO3 30 2.52
    D-Glucose 10 1.802
    Sucrose 140 47.922
    CaCl2 0.3 0.044
    Carbogen needed for pH adjustment final pH 7.3-7.4
    Final osmolarity:
    300-310 mOsm


  3. Potassium gluconate-based intracellular solution (Table 3)


    Table 3. Potassium gluconate-based intracellular solution recipe

    Intracellular solution Concentration in mM g/20 ml
    K-gluconate 130 0.609
    NaCl 6 0.007
    HEPES 10 0.047
    EGTA 0.5 0.004
    Na2-ATP 4 0.044
    Na-GTP 0.35 0.004
    Na2-phosphocreatine 8 0.041
    KOH for pH adjustment final pH 7.2-7.3 Final osmolarity:
    280-290 mOsm

Acknowledgments

This work was supported by grants from the Academy of Finland (1317399), The Finnish National Agency for Education EDUFI, and the Sigrid Juselius Foundation.

The protocol described here was adapted from a previous publication: Nagaeva, E., Zubarev, I., Bengtsson Gonzales, C., Forss, M., Nikouei, K., de Miguel, E., Elsila, L., Linden, A. M., Hjerling-Leffler, J., Augustine, G. J. and Korpi, E. R. (2020). Heterogeneous somatostatin-expressing neuron population in mouse ventral tegmental area.Elife 9: e59328. doi: 10.7554 (Nagaeva et al., 2020).

Competing interests

The authors declare no competing interests.

Ethics

Animal procedures described in the current protocol were authorized by the National Animal Experiment Board in Finland (Eläinkoelautakunta, ELLA; Permit Number: ESAVI/1172/04.10.07/2018).

References

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  2. Cadwell, C. R., Palasantza, A., Jiang, X., Berens, P., Deng, Q., Yilmaz, M., Reimer, J., Shen, S., Bethge, M., Tolias, K. F., Sandberg, R. and Tolias, A. S. (2016). Electrophysiological, transcriptomic and morphologic profiling of single neurons using Patch-seq.Nat Biotechnol 34(2): 199-203.
  3. Franklin, K.B.J. and Paxinos, G. (2008). The Mouse Brain in Stereotaxic Coordinates, 3rd Ed. Elsevier, Amsterdam.
  4. Fuzik, J., Zeisel, A., Mate, Z., Calvigioni, D., Yanagawa, Y., Szabo, G., Linnarsson, S. and Harkany, T. (2016). Integration of electrophysiological recordings with single-cell RNA-seq data identifies neuronal subtypes. Nat Biotechnol 34(2): 175-183.
  5. Gouwens, N. W., Sorensen, S. A., Baftizadeh, F., Budzillo, A., Lee, B. R., Jarsky, T., Alfiler, L., Baker, K., Barkan, E., Berry, K., Bertagnolli, D., Bickley, K., Bomben, J., Braun, T., Brouner, K., et al. (2020). Integrated Morphoelectric and Transcriptomic Classification of Cortical GABAergic Cells. Cell 183(4): 935-953 e919.
  6. Halabisky, B., Shen, F., Huguenard, J. R. and Prince, D. A. (2006). Electrophysiological classification of somatostatin-positive interneurons in mouse sensorimotor cortex. J Neurophysiol 96(2): 834-845.
  7. Ma, Y., Hu, H., Berrebi, A. S., Mathers, P. H. and Agmon, A. (2006). Distinct subtypes of somatostatin-containing neocortical interneurons revealed in transgenic mice.J Neurosci 26(19): 5069-5082.
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  10. Neher, E. and Sakmann, B. (1976). Single-channel currents recorded from membrane of denervated frog muscle fibres. Nature 260(5554): 799-802.
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  12. Sucher, N. J. and Deitcher, D. L. (1995). PCR and patch-clamp analysis of single neurons. Neuron 14(6): 1095-1100.
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  14. Verkhratsky, A. and Parpura, V. (2014). History of electrophysiology and the patch clamp. Methods Mol Biol 1183: 1-19.
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简介

[摘要]电活性细胞(如神经元)的表征需要测量其电特性。由于基因的激活,位置,支配模式的差异,以及功能,在数以百万计的哺乳动物大脑的神经元都在它们的膜特性和能力大大不同来产生动作电位。这些特征可以在全细胞电流钳配置中使用膜片钳技术进行测量,然后对发射模式进行详细的事后分析。这种分析可能很耗时,不同的实验室有自己的方法来执行它,手动或使用自定义编写的脚本。在这里,我们详细描述了一个协议 以腹侧被盖区 (VTA) 神经元中的发射模式配准为例,并介绍一种软件,以便快速方便地进行分析。在这篇文章的帮助下,其他研究小组可以轻松地应用这种方法并生成统一类型的数据,这些数据在大脑区域和各种研究之间具有可比性。

图文摘要:


协议工作流程


[背景]神经元的主要特征是它能够与其他细胞进行快速的化学电交流。在U NIQUE神经元膜构成具有离子通道和其它特定蛋白质的高密度允许的产生的接收足够的输入之后毫秒内的动作电位。因此,如果没有描述其膜的电特性,任何神经元的表征都是不完整的。˚F或这个目的,我们可以使用一个方便的“膜片钳”技术(内尔和萨克曼,1976) ,这是由于在电生理学方法演变的几个世纪(Verkhratsky和Parpura,2014) 。这种方法的一大优点是可以将其与其他现代单细胞方法结合起来,并收集所有必要的数据来定义神经元的类型。通过贴片移液管用细胞内染料填充细胞(Marx等人,2012 年)并在电生理注册后收集细胞的吸出物(Sucher 和 Deitcher,1995 年;Fuzik等人,2016 年)允许同时重建 3D 形态和分析同一细胞的 mRNA 含量和放电模式。有几种类型的膜片钳配置,但我们在这里关注的是全细胞电流钳修改,它允许在控制电流流动的同时记录膜电压的变化;换句话说,它允许注册环动作电位 (AP) 以响应特定的电流注入。

的瓦特的膜片钳技术的空穴细胞电流钳构造是已经在使用了数十年行之有效的方法的电活性的细胞的内在膜性能的表征(内尔和萨克曼,1976;安德鲁,1986;桑切斯-Aguilera等人,2020 年)。尽管大多数研究人员对一组类似的参数感兴趣并使用类似的协议,但实验细节和最终数据是可变的,因此难以比较(参见https://neuroelectro.org/article/index)。虽然准备描述生长抑素表达神经元的小鼠腹侧被盖区(VTA)我们最近的一篇文章(Nagaeva等人,2020年),我们试图收集组最大的膜性能的烧制基于以前的出版物模式分析(Halabisky , 2006; Ma et al ., 2006; Wierenga et al ., 2010) 。这些e 属性的列表可以在Nagaeva等人,2020 年的附录表 1 中找到。类似地,我们做了一个电流-刺激方案,允许记数E-环在一个短期内所有这些性质。此外,我们开发了一个MatLab插件,用于快速方便地提取所有这些参数。

我们的协议本文旨在提供一种用于烧制模式分析清晰的工作流,并且包括从所有步骤的制备急性脑切片的到的最终数据提取。它可以应用于以前未知的神经元的电生理学研究,或作为当前流行的 patch-seq 方法的一部分(Cadwell等人,2016 年;Gouwens等人,2020 年)。对于新描述的神经元,该提取的数据可以被进一步用于神经元亚型根据它们的电膜性能。为此,只需将结果表上传到我们小组先前发布的聚类算法中(进一步参见Nagaeva等人,2020 年;https://github.com/elifesciences-publications/clustering-for-nagaeva-et .-al.-sst-vta)。Ť他的协议将简化在未来的研究烧成图案注册和分析。

关键字:膜片箝, 电流钳, 动作电位, 单细胞电生理学, 急性脑片, 腹侧被盖区, 自动放电模式分析



材料和试剂


急性脑切片的制备
1.烧杯:1 升、2       × 250毫升(VWR,目录号小号:213-1128,213-1124)


2.培养皿60 × 18 mm(VWR,目录号:734-2815p)     

3.用于将碳化物输送到溶液中的管(Ismatec ,目录号:MF0028)     

4.画笔(VWR,目录号:470020-430)     

5.宽颈巴斯德移液器(您可以简单地切断吸头)(Sarstedt ,目录号:NC9891525)     

6.强力胶(乐泰,目录号:230992)     

7.滤纸(Whatman,WHA1001110)     

8.满满一箱铬ü棚冰     

9.带有光滑末端的医用刮刀 150 × 40 × 6 mm(Bochem ,VWR 目录号:231-0601)     

10. Carbogen (95% O 2 + 5% CO 2 ) 

11.脑切片浮网 

一个不错的可以从这里 3D 打印https://3dprint.nih.gov/discover/3dpx-001623。


12.人工脑脊液 (ACSF) 和切割溶液的试剂: 

NaCl (Fisher BioReagents ,目录号:BP358)
KCl (Amresco ,目录号:0395)
MgCl 2 · 6H 2 O(Fisher BioReagents ,目录号:BP214)
NaH 2 PO 4 ·H 2 O(默克,目录号:1.0634)
NaHCO 3 (Sigma-Aldrich,目录号:31437)
D-(+)-葡萄糖(Alfa Aesar ,目录号:A16828)
蔗糖(Fisher Scientific,目录号:10634932)
CaCl 2 (Amresco ,目录号:0556)
 
电生理学
烧杯:250和25毫升(VWR,目录号小号:213-1128,213-1120)
1 毫升微量移液器 ( Thermo Scientific, Finnpipette F2)
用于细胞内溶液的 1.5 毫升 Eppendorf 管(Eppendorf,目录号:0030120086)
带灯丝的玻璃毛细管(World Precision Instruments,目录号:TW150F-4)
画笔(VWR,目录号:470020-430)
用于在显微镜室中固定切片的银/铂丝或特殊网(见图 9)
1毫升注射器(Terumo ,目录号:SS+01T1)
注射器 PVDF Durapore 过滤器(默克密理博,目录号:SLGV013SL)
塑料管 ID 010 × OD 030(Tygon ,目录号:AAD04091)
细胞内溶液 (IS) 的试剂:
K-葡萄糖酸盐(Sigma-Aldrich,目录号:P1847)
HEPES(阿尔法Aesar公司,目录号:A14777)
EGTA(Sigma-Aldrich,目录号:E4378)
Na 2 -ATP(Sigma-Aldrich,目录号:A6419)
Na-GTP(Sigma-Aldrich,目录号:G8877)
Na 2 -磷酸肌酸(Sigma-Aldrich,目录号:P7936)
KOH(Sigma-Aldrich,目录号:221473)
ASCF解决方案(见食谱)
切割溶液(见食谱)
细胞内溶液(见配方)


设备


容量瓶 1 L (品牌,VWR 目录号:612-5082)
大剪刀(Fiskars,目录号:1005151) 
小剪刀(Bochem ,VWR 目录号:233-2121)
小镊子 105 mm(Usbeck ,VWR 目录号:232-0094)
手术刀(Swann- morton ,VWR 目录号:swan0565)
茶匙
用于振动刀的剃刀刀片(World Precision Instruments,目录号:752-1-SS)
同样可以用于程序部分的步骤 A2b中的大脑解剖。


实验室规模(0.001-100 克;梅特勒PJ360 DeltaRange )
pH计(万通,827 pHlab )
磁力搅拌器(默克、IKA大鱿鱼)
实验室水浴(Grant Instruments,Bath JB Aqua 12 Plus)
Vibratome ( Thermo Scientific, Microm HM650V)
渗透压计(Advanced Instruments Inc.,3320 型)
微量移液器拉拔器(Sutter Instruments,P-1000 型)
落射荧光显微镜(奥林巴斯,BX51WI)
荧光光源 – 100 W 带电源的水银弧光灯(Olympus,U-RFL-T)
CCD 相机(索尼,XC-E150)
热控制器(Warner Electric,TC-324B 单通道)
实验室真空泵(KNF、N 811 KTP)
放大器(Molecular Devices、Axon Instruments、Multiclamp 700B)
Digidata (分子设备,Axon 仪器,1322a 型)
显微操作器(Sensapex ,μMp-3)


软件


pClamp 8.2(或更高版本)包(Molecular Devices,https://www.moleculardevices.com/products/axon-patch-clamp-system/acquisition-and-analysis-software/pclamp-software-suite#Resources)
显微镜相机软件
MATLAB R2018b(Mathworks ,https: //se.mathworks.com/products/matlab.html )
Microsoft Excel 2016(微软,https://www.microsoft.com/en-ww/microsoft-365/excel)


程序


急性脑切片的制备
注意:在这里,我们准备了小鼠中脑的水平切片,旨在修补来自 VTA 的dtTomato阳性荧光细胞。阅读器可以使用相同的试剂和程序从任何其他大脑区域制备切片。


如果您使用小于 P30 的幼鼠,请准备人工脑脊液 (ACSF) 和切割溶液(参见配方)。在 4°C 下储存溶液不超过 3-4 天。
如果您在使用Ø lder连续的动物,见亭等。(2018 年)。


将除 CaCl 2以外的所有粉末放入1 升烧杯中,并使用磁力搅拌器与 800 毫升MilliQ水混合。在所有其他粉末完全溶解后加入 CaCl 2 。
倒的溶液倒入1升容量瓶中,并调节音量。调节pH值至7.3 -与卡波冒泡7.4。
为大脑解剖准备您的工作空间。
以方便的顺序放置“干”器械,以便快速到达它们:大剪刀、小剪刀、小镊子、手术刀、弯曲的滤纸片和防水强力胶(图 1)。


图 1.一套“干式”仪器


放乐器 -茶匙,刀片,抹刀医疗用窄,平滑端部,和60毫米培养皿中通过过滤器覆盖纸-和大约250毫升切割在冰上的溶液(图2)。
注意:所述的ACSF溶液应冷却至0℃,和预先具有的冰的浮动块。在手术前和手术期间用carbogen给溶液充气5 分钟(图 2)


提示:锋利且方便的器械可确保任何外科手术的成功。所有器械在使用后都应该用MilliQ水清洗。


˚F igure 2.设置的“冷”的仪器


放一个250毫升烧杯中以不断充气200毫升的ACSF溶液和一个在33℃的水浴中漂浮的净C(图3)。


图 3.装有ACSF 溶液的烧杯和用于在33°C 下孵育脑切片的浮网


准备振动刀:插入刀片并调整程序。我t是方便的具有小的油漆刷,小镊子,和巴斯德吸管与一个宽颈Ñ耳朵的振动切片机(图4)。
注意:我们已经使用了振动切片机程序使用以下参数:进料= 225微米,频率= 88赫兹,振幅= 0.9毫米,和速度= 0.9毫米/秒。


图 4. 用于大脑切片的一组仪器


我们建议您准备好所有必要的设备和仪器,因为您需要在它们之间快速移动。一切准备就绪后,就可以开始大脑解剖了。


脑解剖(约 1 分钟)。
ñ OTES:


一种。由于脑细胞对缺氧非常敏感,在温暖的环境中新陈代谢较快,因此必须尽快将大脑从活体动物转移到冷切割溶液中。我们建议在开始时使用秒表,目标是在 60-80 秒内完成整个过程。     

湾 重要提示:有关在动物斩首前使用的适当麻醉类型的信息,请咨询您当地的动物福利机构。     

C。出于大脑研究的目的,当局有时允许使用斩首方法对新生/幼鼠进行快速物理安乐死,但只能由训练有素的人员执行。     



用大剪刀一步将鼠标斩首。
剪切从皮肤的颈部与眼睛之间用小剪刀。
切小号ķ从回ULL的前囟门沿中线以相同的小剪刀。小心不要用剪刀接触大脑表面。
打开颅骨与小镊子一半一半的中线到了一边。
注:我t是选择非常重要适当的镊子和使用它们仔细,特别是如果你的目标是大脑皮层。

使用手术刀切掉头骨内不必要的大脑部分。我们削减了一半的小脑和额极,如图5。
注意:从这里开始,所有切割程序的目的是获得中脑水平切片,并且应该修改以获得其他感兴趣的大脑区域。


图 5. 内部有大脑的小鼠头部示意图。虚线表示手术刀切口。


注意:在这里,我们切换到“冷”的仪器和环境来减缓新陈代谢。


取出从大脑颅骨与在帮助一个医疗锅铲,并立即将其转移到加气切削液的冷。将其留在那里 1 分钟,同时准备用于强力胶合大脑的冷 vibratome 平台。 
大脑切片(最多 5-10 分钟)。
注:乙在使用振动切片机的ackground知识是必需的牛逼。ο执行大脑切片。


从取出大脑的用茶匙切削溶液,并把它放到所述皮氏培养皿用皮质朝下(即,“上皮层”)。然后,你可以切出一切不必要的部件一个寒冷的刀片,如果你没有服用脑出从之前已经这样做了头骨(见上文)。
在vibratome 平台上放一小滴强力胶,并在弯曲的滤纸的帮助下立即将大脑转移到它上面。不要改变大脑方向(腹侧部分向上)并将其粘合,使小脑面向振动刀刀片。
放置平台到所述缓冲盘和填充与冷充气切割溶液(托盘小号TEP甲3F)。
在小镊子的帮助下,小心地取下仍然粘在大脑上的滤纸。
注意:如果您想延长切片时间,您也可以在缓冲托盘中对切割溶液进行充气。


当大脑如图 6 所示放置时,就可以制作水平中脑切片了。小心地以 225 µm 的步长切掉不必要的上层切片,直到大脑和小脑完全连接。在此级别[对应于来自 Bregma (Franklin and Paxinos, 2008) 的-4.72 ] ,您还将在切片的中心看到一个独特的“圆圈”(参见图 7)。下一个切片是包含 VTA 的第一个中脑切片。


图 6. 振动盘内大脑的方向。该腹侧部分向上和小脑面向刃。所述ř的飞行部分图6示出了托盘内脑的特写。


图 7.开始切割包含 VTA 的水平切片的正确级别(大约在Bregma 的 -4.72 处)。


因此能够获得从小鼠VTA两个或三个225微米厚的水平片段时,其对应于约-4.72毫米,-4.56毫米前囟水平,和-4.28毫米。
传送所得到的片,放入33的帮助下℃的ACSF溶液一巴斯德吸管切割每个切片之后。
将切片在 33°C 下孵育60 分钟;然后,将装有切片的烧杯置于室温下。在S Lices广场是可行的至少4小时。我们在 33°C下孵育60 分钟后立即开始电生理学。
注:这是关键的是发酵过程中切片连续加气,但不围绕浮因为气泡。


电生理学
注:P的电生理和细胞修补技术培训revious理论知识是必需的牛逼Ø进行进一步的协议步骤。


准备基于 K-葡萄糖酸的细胞内溶液 (IS)。
混合用15毫升的所有试剂的的MilliQ水在20毫升烧杯中。
放在冰烧杯中加入ATP,GTP前,和磷酸。
从这里开始,尽量保持该解决方案冷的时候,因为ATP和GTP是对温度敏感。


同时用混合一磁力搅拌器,测量的pH值和调节至7.2,用KOH。
测量渗透压并通过逐毫升添加MilliQ水将其调整到 285 mOsm 。
等分试样的IS在1.5毫升溶液Ë ppendorfs和储存在-20 ℃下的最大2-3个月。
注意:每次在实验前解冻含有 IS的E ppendorf 管,并记住在实验过程中保持低温(在冰上或在 4°C 的冰箱中)。


根据拉马制造商的说明,从硼硅酸盐毛细管中制备 3-5 MΩ 玻璃电极。我们建议使用 4 步程序准备电极。图 8 以我们的Sutter P-1000 拉拔器程序为例。请注意,参数可能会因毛细管的 RAMP测试结果(熔点温度)和拉拔器类型而异。


图 8. Sutter P-1000 拉拔器程序示例,用于制备合适的玻璃电极。请注意斜坡。


准备用于修补的电生理设置。
开关计算机,荧光灯,亮场灯,摄像头,溶液加热控制器,真空泵,放大器,上digidata ,和操纵器。
制备250毫升烧杯中以一个不断充气ACSF溶液。调灌注速度至1-3毫升/分钟为一个1毫升的记录室中。调加热控制器,以保持该溶液在33 ℃。
修补。
转移从片的孵育烧杯中,以该记录室用宽颈巴斯德吸管。
放置在与在其下部的小脑的室的中间的脑切片,如图9所示按该切片下来的银线或专用网片保持器。
注意:脑切片应位于记录室的底部,并在溶液流动过程中保持静止。


图9.方向内的脑切片的所述记录室与朝向研究员小脑。银线将切片固定在腔室底部。


用冷 IS 溶液填充玻璃电极并将其连接到探头。


用于填充定制注射器的图10实施例的细胞内溶液


提示:可以做出很方便注射器是通过使用填充(图10)一1ml注射器中,默克的Millex的Durapore滤波器,和p的柔性塑料管010 ID IECE × OD 030。


通过吹入管并用阀门关闭它,在贴片移液器中产生正压(图 11)。
提示:要检查压力是否足够好,可以打开耳朵附近的阀门。如果压力良好,您应该会听到空心的砰砰声。


图11.经t UBE和阀套被连接到所述探头用于使玻璃电极内部的正/负压力


把电极向下进入该溶液,并检查在移液管的电阻的密封试验窗口(图12)。它应该是 3-5 MΩ。将移液器偏移重置为 0 mV。
注意:从这里到步骤 (v) ,放大器处于电压钳 (VC) 模式。在搜索电池时,电压不会被钳位。



图 12. 密封测试窗口显示 4.3 MΩ的移液器电阻


在小放大倍数下将移液器定位在感兴趣的区域上,例如5 × 。要修补 VTA 中的细胞,请将移液器定位在图 13 中所示的区域上。


图13. VTA区域(标记为绿色虚线左侧)下一个话筒roscope 5 × MAGNIF ication。为了方便起见,在右图像显示标以相同的中脑区域一个内暗虚线的在从-4.72厘米水平脑切片的B regma (富兰克林和Paxinos,2008) 。


用移液器以 4/5机械手速度下降,直到移液器略高于切片焦点。
切换到一个更高的放大倍率(例如,40 × )并找到吸管。
切换到相机视图。
放慢速度机械臂2 - 3/5,慢慢地走下来,对切割面,先用的目标,然后用的吸管。
将移液器停在切片表面正上方。
提示:在此级别,您已经可以评估切片的状况。如果表面有许多气泡细胞,可能很难在切片中找到健康的细胞。

调暗灯光并切换到荧光。搜索一个健康的神经元与该目标。不要移动的目标太远; 否则,将很难再次找到您的移液器。
注:进行u nhealthy神经元是通常为圆形,有清晰可见的细胞核,和一个不清晰,或者有时非常对比轮廓。


找到合适的神经元后,记住它的位置。您可以在屏幕上放置一些标志或以其他方式进行标记(图 14)。 


图 14.放大倍数为 40倍的显微镜下表达荧光标记蛋白的神经元。蓝色十字表示适合修补的神经元。


继续目标并找到移液器。将其移动到切片上方的神经元位置。
吹入管中以确保移液器内有正压。再次将移液器偏移重置为 0 mV。移液器阻力必须在您向下移向神经元时始终保持稳定。
以最慢的机械手速度下到单元格。遵循移液器电阻。
当电阻上升s 0.1-0.2 MΩ 时(您可能还会在细胞上看到一个小黑点,它对应于移液管阴影),通过打开阀门释放移液管压力。同时,使吸管轻微负压吮吸或的帮助下一个注射器。 
此时,电阻上升到 GΩ 值(命名为gigaseal )(图 15),这是贴片成功的第一个迹象。


图 15. 密封测试窗口显示了gigaseal配置(密封电阻 = 2.7 GΩ)


将保持电位钳位在 -70 mV ;通过单击 c-fast 和 c-slow 按钮来消除瞬态电流来补偿快速和慢速电容。保持在细胞千兆欧密封1-2分钟,使的接触更加稳定。
通过使打破隔膜一个强劲,但短期吸力建立全细胞配置。
注意:在这一点上,在电极和神经元成为一个电单元。这允许记录流经神经元通道的电流和/或全细胞膜电压的变化。


断裂后,立即切换到该隔膜测试窗口和读取的晶胞参数(图16)。
注:这是好事,知道了电池的电容(厘米为pF),接入电阻(镭在MΩ) ,和保持电流(保持在pA的)。


Cm可能因电池大小而异,通常在10-100 pF范围内。您可以使用此参数以及从激发模式分析中提取的其他参数来进一步表征神经元。
RA示出之间的接触的质量的玻璃电极和细胞膜; 不应超过 20 MΩ。如果是这样,只需尝试再吸一次并重新制动膜。
保持显示的被保持在细胞所需施加的电流一个一定的电压(毫伏-70在我们的例子); 该值应介于 0 到 -50 pA之间。
切换到所述I = 0模式和读取的在神经元的静息膜电位(RMP)的MultiClamp软件窗口V(毫伏)。
注意:请记下液体接界电位 (LJP),它出现在具有不同离子组成s 的两种液体之间。对于此处使用的细胞外 ACSF 和 IS 解决方案,LJP 为 +12 mV。通常,我们不会在录制过程中更正它,而是总是在“方法”部分提及它。


示出图16.隔膜测试窗口一个在电压钳模式和成功的全细胞构型几个小区参数


切换到的电流钳(IC)模式并运行下面提供的电流的步骤协议(图17)。
ñ OTES:


一种。我们不建议在注入任何电流的电流钳模式,除非你确定的用于正常RMP一个特定的细胞类型。     

湾 e pisodic 刺激协议持续 2 秒。它包括一个 800 毫秒的电流步长,范围从 -60 到 680 pA ,增量为 10 pA,并有 1.2 秒的恢复暂停,以允许细胞恢复到其正常 RMP。此暂停可能会更长,具体取决于细胞类型和刺激强度。     

C。如果您想更精确地定义 AP 阈值和流变碱电流,您可以另外运行一个类似的协议,范围从 0 到 30-50 pA ,增量为 1-pA。     



图 17. 电流步长的推荐方案:800 毫秒电流步长从 -60 pA到 +680 pA ,增量为 10 pA


数据分析


在从原材料*功能。ABF痕迹提取使用FFFPA(快进点火模式分析)在半自动方式,一个开放源代码插件MATLAB。FFFPA提供了灵活的方法来检测所述AP的最常用的参数(包括峰,阈值,以及AHPS)(图18),并自动计算最频繁分析功能,从而允许处理的快速,准确地大批量文件。自动检测的结果是可视化的,如果需要,可以手动检查和调整。Nagaeva et al ., 2020, 附录表 1总结了提取特征的详细定义。


图 18. 动作电位 (AP) 的基本参数,由 FFFPA 插件自动检测。参数定义见Nagaeva et al ., 2020, 附录表 1。


安装


安装需要 MATLAB 2018a 或带有信号处理和曲线拟合工具箱的更新版本。
下载的从FFFPA插件https://github.com/zubara/fffpa并解压牛逼Ø本地目录。
双击的“ fffpa.mlappinstall从MATLAB文件浏览器”文件。
在打开的对话窗口中单击“安装”。
安装完成后,打开从应用的MATLAB应用程序工具条。将出现用于数据导入的图形用户界面(图 19)。


图 19. 用于数据导入的 FFFPA 图形用户界面


数据导入


单击“选择文件”并选择一个或多个'。abf ' 要处理的文件。
指定的单位为Hz的采样频率和发作的时间,以秒为注入电流的步骤的偏移量。
选择要分析的扫描。FFFPA可检测,例如,所述rheobase定义为其中的AP发生第一扫描当前步骤,或在定义为饱和电流步骤的与扫描的接入点的最大数量。
指定了用于汇总统计数据的分析的AP。如果分析了多个扫描,这些选项在第一次扫描和其他扫描之间可能会有所不同。
指定的文件名和所希望的输出格式。


事件检测和特征提取


(可选)进入了“高级”选项卡(图20)指定的功能,你从痕迹希望提取。Nagaeva et al ., 2020, 附录表 1总结了提取特征的详细定义。


图 20. FFFPA 功能选择和高级选项选项卡


单击“主要”选项卡上的“运行”按钮(图 19)。


检测结果的检查和人工修正


要检查自动特征检测的结果,请转到“数据查看器”选项卡(图21)。处理完数据后,单击“运行”后,此选项卡会自动打开。


图 21. FFFPA 数据查看器选项卡


绘图区显示原始跟踪和检测到的事件AP峰(蓝色圆圈),激活阈值(红),后超极化(AHP,青色),和后除极(ADP,未示出)。请注意,在默认情况下,在ADP仅在rheobase第一AP或AP的火车后检测。
使用的“下一步”按钮,通过所有分析的细胞的全部扫描或连续使用的“小区ID”和“扫#”字段手动导航到特定小区的特定横扫。
如果需要,该数据阅读器可以纠正的检测结果。要做到这个,首先点击“编辑,”然后单击要调整,并通过点击跟踪指定它的新位置的元素。要纠正另一点,单击“Z”来激活的再次编辑选项。完成所有更正后,按“X”保存结果。
您还可以通过取消选中“保存到输出”框来从输出中丢弃当前单元格。
对结果满意后,单击“保存选择” 。' 此步骤将根据(调整后的)检测计算特征并将输出文件保存到磁盘。可以在与输入数据相同的目录中找到摘要文件。


笔记


您可以观看一个简短的教程中FFFPA这里插件:https://vimeo.com/497798349。


食谱


注意:所有食谱都应在MilliQ水中制作。


人工脑脊液 (ACSF) 溶液(表 1)


表 1. 人工脑脊液 (ACSF) 溶液配方


ASCF解决方案


以 mM 为单位的浓度


克/升


氯化钠


126


7.36


氯化钾


1.6


0.12


MgCl 2 ·6H 2 O


1.2


0.243


NaH 2 PO 4 ·H 2 O


1.2


0.166


碳酸氢钠3


18


1.5


D-葡萄糖


11


1.98


氯化钙2


2.5


0.37


调节 pH 值所需的碳素


最终 pH 值 7.3-7.4


最终渗透压:


300-310 mOsm


基于蔗糖的切割解决方案(表 2)


表 2. 基于蔗糖的切割溶液配方


切割解决方案


以 mM 为单位的浓度


克/升


氯化钠


60


3.506


氯化钾


2


0.148


MgCl 2 ·6H 2 O


8


1.626


NaH 2 PO 4 ·H 2 O


1.2


0.172


碳酸氢钠3


30


2.52


D-葡萄糖


10


1.802


蔗糖


140


47.922


氯化钙2


0.3


0.044


调节 pH 值所需的碳素


最终 pH 值 7.3-7.4


最终渗透压:


300-310 mOsm


基于葡萄糖酸钾的细胞内溶液(表 3)




表 3. 基于葡萄糖酸钾的细胞内溶液配方


细胞内溶液


以 mM 为单位的浓度


克/20 毫升


葡萄糖酸钾


130


0.609


氯化钠


6


0.007


HEPES


10


0.047


EGTA


0.5


0.004


Na 2 -ATP


4


0.044


Na-GTP


0.35


0.004


Na 2 -磷酸肌酸


8


0.041


用于调节 pH 值的 KOH


最终 pH 值 7.2-7.3


最终渗透压:


280-290 mOsm


致谢


这项工作得到了芬兰科学院 (1317399)、芬兰国家教育局 EDUFI 和 Sigrid Juselius基金会的资助。


该协议描述这里被改编自先前的出版物:Nagaeva ,E.,Zubarev,I.,特松冈萨雷斯,C.,Forss ,M.,Nikouei ,K.,德米格尔,E.,Elsila ,L.,菩提树, AM、Hjerling-Leffler , J.、Augustine, GJ 和Korpi , ER (2020)。小鼠腹侧被盖区中表达生长抑素的异质神经元群。Elife 9:e59328。doi :10.7554 (Nagaeva等人,2020 年)。


利益争夺


作者声明没有竞争利益。


伦理


当前协议中描述的动物程序由芬兰国家动物实验委员会 ( Eläinkoelautakunta , ELLA; 许可证号: ESAVI/1172/04.10.07/2018) 授权。


参考


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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Nagaeva, E., Zubarev, I. and Korpi, E. R. (2021). Electrophysiological Properties of Neurons: Current-Clamp Recordings in Mouse Brain Slices and Firing-Pattern Analysis. Bio-protocol 11(12): e4061. DOI: 10.21769/BioProtoc.4061.
  2. Nagaeva, E., Zubarev, I., Bengtsson Gonzales, C., Forss, M., Nikouei, K., de Miguel, E., Elsila, L., Linden, A. M., Hjerling-Leffler, J., Augustine, G. J. and Korpi, E. R. (2020). Heterogeneous somatostatin-expressing neuron population in mouse ventral tegmental area.Elife 9: e59328.
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