D-serine Measurements in Brain Slices or Other Tissue Explants
脑切片或其他组织外植体中D-丝氨酸测定   

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Neuron
May 2017

 

Abstract

D-serine is an atypical amino acid present in the mammalian body (most amino acids in the mammalian body are L-isomers) that is mostly known in neuroscience for its role as a co-agonist controlling the N-methyl D-aspartate receptor (NMDAR). D-serine levels are decreased in patients with schizophrenia and this is thought to mediate, at least in part, the hypofunction of NMDARs that is central to the glutamate hypothesis for the etiology of this neuropsychiatric disorder. D-serine detection was first established using high performance liquid chromatography, a costly and complex technique that requires high levels of expertise. But with the increasing interest in this unconventional amino acid, there is an increasing need for easier, cheaper and more accessible detection methods. Here we describe the amperometric, biosensor-based method we employed in a recent publication (Papouin et al., 2017b). It allows reliable measurement of D-serine levels from fresh tissue, such as acute brain slices, for concentrations higher than 100 nM, with minimal technical requirements.

Keywords: D-serine (D-丝氨酸), Biosensors (生物传感器), Sarissa Probes (Sarissa探针), NMDA receptor (NMDA受体), Astrocyte (星形胶质细胞), Schizophrenia (精神分裂症), Serine racemas (丝氨酸消旋酶)

Background

The N-methyl D-aspartate receptor (NMDAR) is a receptor for the neurotransmitter glutamate in the brain, spinal cord and in the peripheral nervous system such as enteric neurons. It is also found in renal tubular cells and chondrocytes. In addition to glutamate, the activation of the NMDAR requires the binding of a co-agonist on a dedicated binding site (Johnson and Ascher, 1987; Kleckner and Dingledine, 1988). The unconventional amino acid D-serine is the endogenous co-agonist of the NMDAR in numerous regions of the nervous system (see Papouin et al., 2017a). It is also found abundantly in the liver and kidneys where its degradation and excretion take place (Montesinos Guevara and Mani, 2016). D-serine is also found in the gut, where its function and origin (host metabolite or bacterial origin) are unclear. Therefore, detecting and measuring D-serine levels has become necessary in several subfields of neuroscience and other disciplines but has proven technically challenging. In the brain and spinal cord, assessing the occupancy of the NMDAR co-agonist binding site is an excellent first approach to assess D-serine levels (Papouin et al., 2012; Papouin et al., 2017b; Ferreira et al., 2017). However, major limitations of this approach are that 1) it provides little quantitative insights into the actual concentration of D-serine, 2) it is subject to a strong ceiling effect (once the co-agonist binding site is saturated, higher levels of D-serine go undetected), 3) glycine can also bind to the NMDAR co-agonist binding site and compete with D-serine, 4) this approach is subject to changes and differences in the affinity of the NMDAR co-agonist binding site, and finally 5) this method is only useful in conditions where recording NMDAR activity is technically possible. Therefore, obtaining direct measurements of D-serine has become a necessity and challenge. Two methods are currently available. The first one is electrophoresis-based, in particular high performance liquid chromatography (Papouin et al., 2017b) or capillary electrophoresis (Ferreira et al., 2017) which has been amply documented. While they provide high levels of precision and reliability, they also require expensive equipment and extensive technical expertise. The second one is based on the use of biosensors such as those developed by Sarissa (Dale et al., 2005) or by several independent labs such as Pernot et al., 2008, which requires minimal equipment or technical expertise. Biosensors usually consist of probes coated with the enzyme D-amino acid oxidase which degrades D-serine to produce electrons. They function as amperometric probes, where electrical current produced during D-serine degradation is used to measure the amount of D-serine present. We found that the use of sensors comes with pitfalls and caveats that, if not carefully avoided or controlled, can lead to aberrant measurements. Therefore, in a recent study (Papouin et al., 2017b) we developed a protocol to reliably detect D-serine levels in brain slices using D-serine biosensors from Sarissa. Here, we describe this protocol in greater detail, and in a step-by-step manner. This protocol is based on obtaining conditioned medium from brain slices and, therefore, can be easily adapted to any tissue of interest, such as the spinal cord, kidney or liver, provided that acute slices can be obtained.

Materials and Reagents

Materials

  1. For the ‘nest Beaker’
    1. Nylon tights
    2. Instant superglue (such as Scotch Super Glue, 3M, catalog number: AD124 )
    3. 15 ml tubes (such as VWR, catalog number: 89039-670 US, 525-0450 Europe)
    4. Disposable 6 cm diameter plastic Petri dish (such as Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 123TS1 )

  2.  For dissection
    1. Large kitchen scissors or guillotine
    2. Straight fine scissors (such as Fine Science Tools, catalog number: 14060-11 )
    3. Curved spatula (such as Fine Science Tools, catalog number: 10092-12 )
    4. Scalpel (such as Fine Science Tools, catalog number: 91003-12 )
    5. Glass disposable Pasteur pipet (such as Fisher Scientific, FisherBrand, catalog number: 13-678-6A )
    6. Dropper bulb (such as Fisher Scientific, FisherBrand, catalog number: 03-448-25 )
    7. Plastic container, about 2.5 cm high and 150 ml, such as the lid of a pipet tip box or a large glass Petri dish (Cole-Parmer Instrument, catalog number: EW-34551-06 )
    8. Whatman paper (GE Healthcare, Whatman, catalog number: 1001-090 )
    9. Disposable Razor blade (such as Personna Double Edge Razor Blades [Amazon, PERSONNA, catalog number: BP9020 ])

  3. For conditioned medium procedure
    1. Disposable 6 cm diameter (or larger) plastic Petri dish (such as Thermo Fisher, catalog number: 123TS1 )
    2. 1.7 to 2 ml microtubes (such as Sorenson BioScience, catalog number: 16070 )

  4. For biosensors holders
    1. 2 ml Falcon pipette (Fisher Scientific, catalog number: 13-678-11C )
    2. Heat-Shrink tubes (from RadioShack)


Reagents

  1. Glucose (Sigma-Aldrich, catalog number: G7021 )
  2. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  3. Sodium phosphate monobasic anhydrous (VWR, catalog number: 470302-666 )
    Manufacturer: ALDON, catalog number: SS0756-500GR .
  4. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S5761 )
  5. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
  6. Magnesium chloride solution (1 M) (Sigma-Aldrich, catalog number: 63069 )
  7. Calcium chloride solution (1 M) (Sigma-Aldrich, catalog number: 21115 )
  8. D-serine (Sigma-Aldrich, catalog number: S4250 )
  9. Stock artificial cerebrospinal fluid (ACSF) solution (see Recipes)
  10. Ice-cold Slicing ACSF stock solution (see Recipes)
  11. Recovery ACSF stock solution (see Recipes)
  12. Experimental ACSF stock solution (see Recipes)

Equipment

  1. 250 ml Pyrex beaker (such as VWR, catalog number: 10754-952 )
  2. Straight spring scissors (such as Fine Science Tools, catalog number: 15018-10 )
  3. Curved fine forceps (such as Fine Science Tools, catalog number: 11152-10 )
  4. 600 ml Pyrex beaker (such as VWR, catalog number: 10754-956 )
  5. 95% O2/5% CO2 tank (such as AirGas, catalog number: Z02OX9522000043 )
  6. Vibratome (such as Leica, model: Leica VT 1200 S , catalog number: 14048142066)
  7. Bath heater (such as Thermo Fisher Scientific, Thermo Scientific, model: Precision 180 , catalog number: 51221073)
  8. Digi IVY DY2023 Bipotentiostat (Digi-Ivy, model: DY2023 ) (www.digi-ivy.com)
  9. Connecting cables (provided with bipotentiostat)
  10. Computer connected to the bipotentiostat
  11. Sarissa Probe D-serine biosensor (Sarissa Biomedical, catalog number: SBS-DSER-05-50 )
  12. Sarissa Probe Null Sensor (Sarissa Biomedical, catalog number: SBS-NUL-05-50 )
  13. Minimal components of a classic electrophysiology rig (Figure 1):
    Perfusion/immersion chamber (such as Warner Instruments, model: RC-26G , catalog number: 64-0235)


    Figure 1. Setup needed to mount the biosensors (see text). For clarity, there is no liquid perfused through the chamber on the above image and only one biosensor was mounted.

  14. Custom-made perfusion line (gravity or pump-operated) using flexible plastic tubing (such as Cole-Parmer Instrument, Masterflex, catalog numbers: ZM-96400-13 and ZM-96400-14 )
    Note: If the perfusion system is gravity-based, a vacuum line connected to the exit end of the chamber will be required to suction the liquid from the chamber and maintain a steady state immersion level in the chamber.
    1. Chamber temperature controller (such as Warner Instruments, model: TC-344C , catalog number: 64-2401)
    2. Microscope or any time of magnifying binocular allowing a 5x magnification (and lighting)
    3. Manual manipulators to allow the placement of the sensors (such as NARISHIGE, model: M-152 )
    4. Home-made connector to plug the sensors to the cables provided with the bipotentiostat and allowing placement in the manipulators (see Figure 2)
    5. Biosensor holder. Using a 2 ml Falcon pipette, cut the desired length (we recommend 10 cm), insert the biosensor connecting cable, wrap in heat-shrink tube (from RadioShack) as shown in Figure 2 and gently heat with a lighter or a soldering iron


      Figure 2. Making a biosensor holder. This will help manipulation and placement of the biosensors with manual manipulators.

Software

  1. DY2000EN software (provided with bipotentiostat)
  2. Clampfit (version 9.2 minimum)
    Note: If experiments are run on an electrophysiology set-up. Note that the DY2000EN software provides the sufficient analytical capabilities, but they were not used in our protocol.

Procedure

Before starting:
This protocol requires obtaining acute brain slices or acute slices from other tissue explants. Refer to Bio-protocol ‘Obtaining Acute Brain Slices’ (Papouin and Haydon, 2018) for a full and detailed description of that procedure. The present protocol requires the use of some material also described in Bio-protocol ‘Obtaining Acute Brain Slices’, such as a ‘nest beaker’ (Figure 3) and modified Pasteur pipette dropper (Figures 3 and 4). For a full description and instructions on how to build these, please refer to Bio-protocol ‘Obtaining Acute Brain Slices’.
1. Nest beaker
2. Modified Pasteur pipette dropper


Figure 3. Nest beaker


Figure 4. Modifier Pasteur pipette dropper. Break the thinnest end of a glass disposable Pasteur pipet, and insert that end in a Dropper bulb.

Setting up:

  1. Prepare 1 L of stock ACSF the day prior and store overnight at 4 °C (see Recipes).
  2. On the day of the experiment, prepare 300 ml of ice-cold slicing ACSF (see Recipes) for acute brain slicing and refer to Bio-protocol ‘Obtaining Acute Brain Slices’ for a detailed procedure on how to obtain brain slices.
  3. Prepare 150 ml of recovery ACSF in the ‘nest beaker’ as instructed in Bio-protocol ‘Obtaining Acute Brain Slices’.
  4. Reserve the rest of the ACSF (~550 ml) to use as ‘Experimental ACSF’ (see Recipes)


  1. Obtain brain slices (or explants of the tissue of interest)
    See Bio-protocol    ‘Obtaining Acute Brain Slices’ Papouin and Haydon (2018). This is a very classic procedure used routinely in many labs for in vitro electrophysiology and various descriptions can be found in textbooks, on PubMed (for instance, Ting et al., 2014), or on Jove website (https://www.jove.com/video/2330).

  2. Incubate slices to obtain conditioned medium
    1. In the case of the dorsal hippocampus, typically 6-8 hemi-slices should be obtained from an adult male mouse. This may vary depending on your brain region or tissue of interest. Once the recovery is over (Papouin and Haydon, 2018), transfer hemi-slices individually in a Petri dish filled with room temperature oxygenated recovery ACSF (from the nest beaker–a few ml will suffice), and carefully separate the hippocampus from the rest of the slice with spring scissors and fine forceps (Figure 5A). To minimize manipulation and damage to slices, a small portion of cortex immediately above the hippocampus (motor and somatosensory cortex typically) can be left attached (it would therefore participate in the following incubation and conditioned medium preparation). Transfer the ‘isolated’ hippocampal slices back to the nest beaker and allow an additional 15 min of recovery. This hippocampus isolation procedure can be greatly facilitated by removing the oxygenation tube from the Petri dish, on the condition that the procedure only takes 1 or 2 min and that slices are immediately transferred back to the nest beaker.


      Figure 5. Incubation of hippocampal slices to obtain conditioned medium. A. In a Petri dish containing recovery ACSF, separate the hippocampus from the rest of the slice with spring scissors and fine forceps. B. After briefly transferring slices into experimental ACSF, transfer them into a 1.7 ml microtube filled to 1.5 ml with RT experimental ACSF. Oxygenate gently and leave in a tube rack for 90 min.

    2. Using the modified Pasteur pipette (Figure 4), gently transfer all slices in a small Petri dish containing oxygenated experimental ACSF (see Recipes) at RT. Make sure the slices are not swirling around because of the oxygenation. This step is only required to transfer slices from recovery ACSF to conditioning ACSF (Step B3).
    3. After a few seconds, transfer 3 to 4 hippocampal slices at the bottom of a 1.7 ml microtube with the modified Pasteur pipette, and fill the tube up to 1.5 ml with RT experimental ACSF. Insert oxygenation tubing and gently oxygenate (Figure 5B). Reduce the rate of oxygenation so that the content of the tubes is not splashing and so that the slices are not swirling. Slices should remain at the bottom of the tube, gently waving. Incubate for 90 min at RT on a tube rack, while regularly checking that the oxygenation is appropriate.
    4. Carefully transfer the conditioned medium (CM) with a 1 ml pipette into a clean microtube and immediately freeze at -80 °C until used for D-serine measurement. If used at the same day, store at 4 °C. Be careful not to pipette any tissue with the CM. It is safer to leave a few µl in the tube. Unless they are treated differently, pool together the CM from all slice incubations in a 15 ml tube. The remaining experimental ACSF (i.e., vehicle ACSF) should be stored along with the CM samples, at -80 °C or 4 °C. Freeze the incubated tissue left at the bottom of the tubes at -80 °C until protein extraction and optic density measurement is performed with a Pierce BCA protein assay kit (not described in this protocol).

  3. Prepare biosensors
    This protocol was designed for the use of Sarissa probes. It can easily be adapted for other D-serine sensors. If using Sarissa D-serine probes, prepare the biosensors according to manufacturer’s instructions Sarissa Biomedical (see also Dale et al., 2005) or as follows:
    1. Run the vehicle ACSF (same ACSF that was used to obtain CM from the hippocampal slices) through the electrophysiology chamber and turn the chamber heater to 33 °C.
    2. Once the flow is steady and desired temperature is reached, mount the D-serine and the Null sensor probes on the biosensor-holder attached to the micromanipulators (Figure 1).
    3. Using the micromanipulators, submerge the probes in the recording chamber and position them so that they are close to each other and at the same depth (Figures 1 and 6). Preferably, the two probes should be facing each other, at a similar angle respective to the flow. Let sensors rehydrate for 15-30 min. Note that while rehydrating, the sensors will swell in size (like a sponge). Give the sensors enough space to rehydrate without coming into contact with one another or the bottom of the chamber. The probe part of the biosensors is extremely fragile: make sure it does not come into contact with anything that could damage it.


      Figure 6. Biosensor probe submersion and positioning in the recording chamber
      Notes: Before starting ‘D- Run conditioned medium on biosensors’ make sure the perfusion line is as short as possible. Make sure the perfusion rate is no greater than 0.5 ml/min. Make sure that the sensor part of the probe is constantly submerged. Once the probes are rehydrated, they cannot be exposed to the air more than a few seconds. Exposing the sensors to the air while polarized at +500 mV will result in immediate and irreversible damage to the sensor. We strongly recommend running a trial run with dummy sensors.

    4. Using the bipotentiostat and the polarity command on the software interface, manually polarize the sensors at +500 mV and switch the polarity to -500 mV and back, cycling back and forth about 10 to 12 times (see Figure 7).
    5. Allow polarizing to +500 mV for at least 60 min or until capacitive decay is imperceptible over the course of a 15-20 min ‘run’ (Figure 7).


      Figure 7. Print screen of a calibration experiment (10 μM D-serine) run on a D-serine sensor (top red trace) and null sensor (bottom blue trace) using the DY2000 software. Note the parameters used, in particular, sampling rate: 50 Hz, Duration of a run: 1,000 sec, Sensors potential: 0.5 V and A/V scale factors. Note that at the end of each run, the software will show a ‘Ready…’ signal (bottom right-hand corner), signaling that a new run can be started (by pressing the RUN button, bottom right-hand corner). However, the run that just ended is not automatically saved. Make sure to press Save (bottom left, green button) prior to starting another run or data will be lost. Also note that ‘noise’ artifacts typically appear on both traces. However, D-serine perfusion elicits no current on the null sensor. In fact, one can still detect a ~20 pA capacitive decay over the course of 1,000 sec recording on the null sensor trace.

  4. Run conditioned medium on biosensors
    1. Thaw the conditioned media on ice and then leave at RT. Tubes may contain some deposit. Do not perfuse the deposit through the line, instead pipette out the CM into a clean tube. Quickly switch the perfusion line from the vehicle solution to the tube containing the CM. Make sure there are no bubbles in the line (we recommend ‘pinching’ the perfusion line when switching solutions).
    2. Once the volume of CM runs through, switch the line back to the vehicle solution for as long as necessary to obtain a full wash-in/wash-out curve (Figure 8) and to clean the line of residual D-serine before the next measurement.
    3. If the next CMs to be run on the sensors were obtained on a different day using different vehicle ACSF, run the corresponding vehicle solution for at least 15 min before running the next CM. Make sure the sensors are not exposed to the air during this process.
      Note: The DY2000 software provided with the biopotentiostat requires saving the results from each run after the run is complete. It is therefore critical to remember to click SAVE at the end of each induvial run (Figure 7).
    4. It appears that different sensors tend to give very different reading qualities and steadiness. We recommend proceeding to Procedure D only once, it is ascertained that the set of biosensors provides a steady and clean amperometric signal devoid of artifacts and fluctuations due to perfusion flow or electrical noise. In our study, we chose to abort or exclude all experiments in which the baseline behavior of the sensors would prevent accurate and reliable evaluation of D-serine levels in CM.


      Figure 8. Print screen of the Clampfit analysis of a CM D-serine measurement. Top trace: D-serine sensor. Middle trace: Null sensor. Bottom trace: D-serine sensor minus null sensor. Cursors 1-2 show where the baseline subtraction is made so that the baseline portion of the trace reads 0 pA. Cursors 3-4 show where the mean peak response is measured.

  5. Calibrate the biosensors
    1. In advance, prepare standard solutions of D-serine of, for example, 0.05, 0.5, 5 and 50 µM using serial dilutions in vehicle ACSF. We recommend increasing the accuracy of the calibration around the concentrations of D-serine you expect to detect in the CM.
    2. Switch from the vehicle solution to the D-serine standard solution, beginning with the lowest concentration standard.
    3. Run each of the D-serine standard solutions through the line until a plateau is reached (Figure 9) before switching back to vehicle ACSF. Allow sufficient time between each solution to thoroughly and completely wash-out after each application (this can take up to 20 min for the highest concentrations depending on your set-up).
      Notes: For Procedures D and E, when switching from one solution to the other, make sure no interruption in the perfusion flow occurs to guarantee that the amount of liquid (and liquid flow) remains constant in the chamber. Proceeding otherwise could compromise the immersion of the sensors and expose and irreversibly damage them.


      Figure 9. Print screen of the Clampfit analysis of a 1 μM D-serine calibration experiment. Top trace: D-serine sensor. Middle trace: Null sensor. Bottom trace: D-serine sensor minus null sensor. Cursors 1-2 show where the baseline subtraction is made so that the baseline trace reads 0 pA. Cursors 3-4 show where the mean peak response is measured. Artifacts were intentionally created on traces around the start of perfusion of the 1 μM D-serine solution and after ~3 ml of perfusion. This type of experiment was used to determine the minimal volume of liquid required to measure D-serine concentration in CM reliably.

  6. Discard or store the biosensors
    1. Make sure that the biosensors are no longer polarized and the bipotentiostat is turned off before handling and unplugging the biosensors.
    2. If sensors are to be reused, follow manufacturer’s instructions. Quickly transfer the sensors to Buffer A (see manufacturer’s instructions) in a rehydration pot at 4 °C and place in a fridge. Alternatively, from our experience, we found that if handled properly, sensors can be kept overnight in cold ACSF (4 °C) and reused at least once within the next 48 h without major loss of sensitivity.

Data analysis

The data analysis can be run using Clampfit, like any electrophysiology trace (see Figures 8 and 9). However, this requires exporting the data as an Excel file beforehand. Follow these quick simple steps to do so.

  1. Right-click on the file to be analyzed.
  2. Open with Excel.
  3. Delete the first rows that contain details about the experiment.
  4. Optional: Delete the time column if you know the sampling rate. Add an additional column that corresponds to the signal from the null sensors subtracted from that of the D-serine sensor (D-ser–Null sensor). Note that this subtraction can also be done on Clampfit.
  5. Save as a separate text file (.txt).
  6. Open that text file with Clampfit.
    Note: Clampfit will ask the number of signals, sampling rate, units, scale, and time frame. Give pertinent information with regard to point #4 above.
  7. To obtain the D-serine–Null sensor trace, if this was not done on the Excel text file, save each trace individually (as a D-serine and a Null trace) and, while both files are open simply open the tab Analyze > subtract control. The D-serine–Null sensor trace will appear as a separate individual trace and can be saved as a separate file.
  8. Using Clampfit cursors and tools, zero the trace at baseline and measure the peak response of the D-serine–Null sensor trace. We recommend measuring the average current value (in pA) over one or several minutes of the plateau response for more accuracy, instead of simply measuring the ‘absolute peak’ which might give false values given that traces are usually noisy (Figures 8 and 9).
  9. D-serine biosensors have a specified lower detection limit of 100 nM. However, in our hands, they successfully detected concentrations as low as 50 nM (when running D-serine standards). It is up to the experimenter’s discretion to decide whether D-serine measurements falling below the specified detection limit should be excluded or whether they should be considered accurate. In our study (Papouin et al., 2017b), we chose to be conservative and show such data points but exclude them from the overall analysis.

Notes

  1. The volume of CM incubated with slices (3 ml in this case) should be set by preliminary experiments aimed at determining the minimal volume of CM that can be run through the perfusion system that allows plateau detection by the biosensors (see Figure 9 and Legend).
  2. Though no current is generally observed on the Null sensor, currents measured on this sensor electrode must be subtracted from currents measured on the D-serine sensor electrode to obtain a pure ‘D-serine-induced’ amperometric signal; or ‘net D-serine signal’.
  3. Sensors should be calibrated at the end of every experiment to measure their sensitivity. For the first few experiments, we recommend calibrating the sensors before running the CM. This will allow inexperienced experimenters to familiarize themselves with the procedure. This also presents the advantage that if any problem were to occur over the course of the experiments (such as a perfusion issue and damage to the biosensors), it will still be possible to determine the absolute concentration of D-serine in all CM run until then. However, it is unknown whether running high concentrations of D-serine (50-100 µM) for several minutes at a time causes any loss of DAAO activity or integrity, and whether, therefore, this causes a loss of sensitivity of the sensors. Since D-serine levels in the CM are typically low, this could result in a lack of detection or underestimation in the following D-serine measurements. One also runs the risk of residual subthreshold amounts of D-serine being present in the line or chamber and false detection during the subsequent CM perfusion.
  4. Classical use of these biosensors normally consists of penetrating brain slices with the biosensor electrode themselves (Shigetomi et al., 2013). We strongly advise against using this method as it will inevitably result in an immediate peak detection of tens of mM of D-serine, due to tissue destruction and irreversible damage to cells, causing intracellular D-serine (which accounts for 99.9% of brain D-serine) to be released into the extracellular space. After tens of minutes of decay, this is expected to stabilize to a steady-state level thought to reflect the endogenous extracellular concentration of D-serine in slices. Instead, this is more likely to reflect residual amounts of D-serine escaping the scarring/dying tissue surrounding the biosensor. Additionally, this approach is highly dependent on how deep sensors are forced into the slice, which dictates how much of the sensor is in direct contact with damaged tissue and therefore how much D-serine is detected. Last but not least, we found that such a method could cause deterioration of the sensor itself upon penetration in the slice. Together these concerns encouraged us to employ a different approach described in this protocol.

Recipes

  1. Stock artificial cerebrospinal fluid (ACSF) solution (1 L, store at 4 °C)
    Glucose 10 mM (1.8 g for 1 L)
    Sodium chloride 125 mM (7 g for 1 L)
    Sodium phosphate monobasic anhydrous 1.25 mM (0.119 g for 1 L)
    Potassium chloride 2.5 mM (0.23 mg for 1 L)
    Sodium bicarbonate 26 mM (2.18 mg for 1 L)
    Adjust pH 7.3 and osmolarity 290-300 mOsm L-1
    Make up to 1 L with ddH2O
  2. Ice-cold slicing ACSF (~300 ml)
    Stock ACSF
    2 mM magnesium chloride
    1 mM calcium chloride
  3. Recovery ACSF (~150 ml)
    Stock ACSF
    1.5 mM magnesium chloride
    2 mM calcium chloride
  4. Experimental ACSF (~550 ml)
    Stock ACSF
    1.3 mM magnesium chloride
    2 mM calcium chloride
    Note: Of which ~3 ml will be used to obtained hippocampal slices-conditioned medium (conditioning ACSF) and the rest will be used as a ‘blank’ during the amperometric D-serine measurements (vehicle ACSF). Label and store at RT for < 2 days or freeze at -20 °C. Each conditioned medium must have its corresponding vehicle ACSF.

Acknowledgments

This work was supported by two Philippe Foundation grants and a Human Frontier Science Program long-term fellowship (LT000010/2013) awarded to T.P., and two NIH/NINDS R01 grants (NS037585 and AA020183) awarded to P.G.H. who is also the founder of GliaCure. Authors declare no conflict of interest. We thank Jaclyn M. Dunphy for her careful proofreading of this protocol.

References

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  12. Ting, J.T., Daigle, T.L., Chen, Q., Feng, G. (2014). Acute brain slice methods for adult and aging animals: application of targeted patch clamp analysis and optogenetics. Methods Mol Biol 1183: 221-42.

简介

D-丝氨酸是存在于哺乳动物体内的非典型氨基酸(哺乳动物体内的大部分氨基酸是L-异构体),其在神经科学中主要是作为控制N-甲基D-天冬氨酸受体的共激动剂的作用而已知的NMDA受体)。精神分裂症患者的D-丝氨酸水平降低,这被认为至少部分地介导了NMDARs功能减退,这种谷氨酸假说对于这种神经精神障碍的病因至关重要。 D-丝氨酸检测首先使用高效液相色谱法建立,这是一种昂贵且复杂的技术,需要高水平的专业知识。但随着对这种非常规氨基酸的兴趣日益增加,人们越来越需要更容易,更便宜和更容易获得的检测方法。在这里,我们描述了我们在最近的出版物(Papouin等人,2017b)中采用的安培法,基于生物传感器的方法。它可以对新鲜组织的D-丝氨酸水平进行可靠的测量,如急性脑切片,浓度高于100 nM,技术要求最低。

【背景】N-甲基D-天冬氨酸受体(NMDAR)是脑,脊髓和周围神经系统如肠神经元中神经递质谷氨酸的受体。在肾小管细胞和软骨细胞中也有发现。除谷氨酸外,NMDAR的激活需要在专用结合位点上结合共激动剂(Johnson和Ascher,1987; Kleckner和Dingledine,1988)。非常规氨基酸D-丝氨酸是神经系统众多区域中NMDAR的内源性共激动剂(参见Papouin等人,2017a)。在肝脏和肾脏中也可以发现其丰富的降解和排泄(Montesinos Guevara and Mani,2016)。 D-丝氨酸也存在于肠道中,其功能和来源(宿主代谢物或细菌来源)不清楚。因此,检测和测量D-丝氨酸水平在神经科学和其他学科的几个子领域中已经成为必要,但是在技术上已经证明具有挑战性。在脑和脊髓中,评估NMDAR共激动剂结合位点的占有率是评估D-丝氨酸水平的极好的第一种方法(Papouin等人,2012; Papouin等人。2017b; Ferreira et al。,2017)。然而,这种方法的主要限制是:1)对D-丝氨酸的实际浓度几乎没有定量的认识; 2)它受到强烈的上限效应(一旦共激动剂结合位点饱和,则更高水平的D未检测到丝氨酸),3)甘氨酸还可以结合NMDAR共激动剂结合位点并与D-丝氨酸竞争,4)该方法经历NMDAR共激动剂结合位点亲和力的变化和差异,以及最后5)这种方法仅适用于记录NMDAR活动在技术上可行的情况。因此,直接测量D-丝氨酸已成为必要和挑战。目前有两种方法可用。第一种是基于电泳的,特别是高效液相色谱(Papouin等人,2017b)或毛细管电泳(Ferreira等人,2017),其已经充分记录。虽然它们提供高水平的精度和可靠性,但也需要昂贵的设备和广泛的技术专长。第二个是基于使用生物传感器,例如由Sarissa(Dale等人,2005)开发的那些生物传感器,或者由几个独立的实验室,如Pernot等人, 2008年,这需要最少的设备或技术专长。生物传感器通常由涂有D-氨基酸氧化酶(其降解D-丝氨酸以产生电子)涂覆的探针组成。它们起安培计探针的作用,其中在D-丝氨酸降解过程中产生的电流用于测量存在的D-丝氨酸的量。我们发现传感器的使用会带来一些陷阱和警告,如果不仔细避免或控制,会导致异常的测量。因此,在最近的研究中(Papouin等人,2017b),我们开发了使用来自Sarissa的D-丝氨酸生物传感器可靠地检测脑切片中的D-丝氨酸水平的方案。在这里,我们更详细地描述了这个协议,并且一步步地描述了这个协议。该协议基于从脑切片中获得条件培养基,因此可以容易地适应于任何感兴趣的组织,例如脊髓,肾脏或肝脏,只要可以获得急性切片。

关键字:D-丝氨酸, 生物传感器, Sarissa探针, NMDA受体, 星形胶质细胞, 精神分裂症, 丝氨酸消旋酶

材料和试剂

的材料

  1. 对于“巢烧杯”
    1. 尼龙紧身衣
    2. 即时超强胶(如苏格兰超级胶,3M,目录号:AD124)
    3. 15毫升管(如VWR,目录号:89039-670美国,525-0450欧洲)
    4. 一次性6厘米直径的塑料培养皿(如Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:123TS1)

  2. &nbsp;解剖
    1. 大厨房剪刀或断头台
    2. 直的罚款剪刀(如精细科学工具,目录号:14060-11)
    3. 弯曲的铲子(如Fine Science Tools,目录号:10092-12)
    4. 手术刀(如Fine Science Tools,目录号:91003-12)
    5. 玻璃一次性巴斯德吸管(如Fisher Scientific,FisherBrand,目录号:13-678-6A)
    6. 滴管灯泡(如Fisher Scientific,FisherBrand,目录编号:03-448-25)
    7. 塑料容器,高约2.5厘米,150毫升,如吸管尖端盒或大玻璃培养皿的盖子(Cole-Parmer Instrument,目录号:EW-34551-06)
    8. Whatman纸(GE Healthcare,Whatman,目录号:1001-090)
    9. 一次性剃刀刀片(例如Personna双刃剃刀刀片[亚马逊,PERSONNA,目录号:BP9020])

  3. 对于条件培养基程序
    1. 一次性6厘米直径(或更大)塑料培养皿(如Thermo Fisher,目录号:123TS1)
    2. 1.7至2ml微管(例如Sorenson BioScience,目录号:16070)

  4. 对于生物传感器持有人
    1. 2ml Falcon移液管(Fisher Scientific,目录号:13-678-11C)
    2. 热缩管(来自RadioShack)


的试剂

  1. 葡萄糖(Sigma-Aldrich,目录号:G7021)
  2. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  3. 无水磷酸二氢钠(VWR,目录号:470302-666)
    制造商:ALDON,产品目录号:SS0756-500GR。
  4. 碳酸氢钠(NaHCO 3)(Sigma-Aldrich,目录号:S5761)
  5. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
  6. 氯化镁溶液(1M)(Sigma-Aldrich,目录号:63069)
  7. 氯化钙溶液(1M)(Sigma-Aldrich,目录号:21115)
  8. D-丝氨酸(Sigma-Aldrich,目录号:S4250)
  9. 股票人造脑脊液(ACSF)的解决方案(见食谱)
  10. 冰冷切片ACSF储备溶液(见食谱)
  11. 恢复ACSF储备溶液(见食谱)
  12. 实验ACSF原液(见食谱)

设备

  1. 250毫升派热克斯烧杯(如VWR,目录号:10754-952)
  2. 直弹簧剪刀(如Fine Science Tools,目录编号:15018-10)
  3. 弯曲细镊子(如Fine Science Tools,目录号:11152-10)
  4. 600毫升派热克斯烧杯(如VWR,目录号:10754-956)
  5. 95%O 2/5%CO 2罐(例如AirGas,目录号:Z 0 OXX9522000043)。
  6. 颤音(如Leica,型号:Leica VT 1200 S,目录号:14048142066)
  7. 浴加热器(如Thermo Fisher Scientific,Thermo Scientific,型号:Precision 180,目录号:51221073)
  8. Digi IVY DY2023 Bipotentiostat(Digi-Ivy,型号:DY2023)( www.digi-ivy.com
  9. 连接电缆(提供bipotentiostat)
  10. 电脑连接到bipotentiostat
  11. Sarissa探针D-丝氨酸生物传感器(Sarissa Biomedical,目录号:SBS-DSER-05-50)
  12. Sarissa探针空传感器(Sarissa Biomedical,目录号:SBS-NUL-05-50)
  13. 经典电生理设备的最小组件(图1):
    灌注/浸泡室(如华纳仪器,型号:RC-26G,目录号:64-0235)


    图1.安装生物传感器所需的设置(参见正文)。为了清楚起见,在上图中没有通过腔室灌注液体,只安装了一个生物传感器。

  14. 使用柔性塑料管(如Cole-Parmer Instrument,Masterflex,目录号:ZM-96400-13和ZM-96400-14)定制的灌注线(重力或泵操作)
    注:如果灌注系统是基于重力的,则连接到腔室出口端的真空管线将需要从腔室抽吸液体并保持腔室中的稳定状态浸没水平
    1. 室温控制器(如华纳仪器,型号:TC-344C,目录号:64-2401)
    2. 显微镜或放大双目镜的任何时间允许放大5倍(和照明)
    3. 手动操纵器允许放置传感器(如NARISHIGE,M-152型)
    4. 自制连接器将传感器插入双定压器附带的电缆,并允许放置在操纵器中(见图2)
    5. 生物传感器支架。如图2所示,使用2 ml Falcon移液管,剪下所需长度(推荐10 cm),插入生物传感器连接电缆,包裹在热收缩套管(RadioShack公司制造)中,然后用打火机或烙铁轻轻加热< br />

      图2.制作生物传感器支架这将有助于使用手动操纵器来操作和放置生物传感器

软件

  1. DY2000EN软件(提供bipotentiostat)
  2. Clampfit(最低版本9.2)
    注意:如果实验在电生理学设置上进行。请注意,DY2000EN软件提供了足够的分析功能,但它们并未在我们的协议中使用。

程序

开始之前:
该协议需要获得急性脑切片或来自其他组织外植体的急性切片。请参阅Bio-protocol“获取急性脑切片”(Papouin和Haydon,2018年),了解该程序的完整详细描述。目前的方案需要使用Bio-protocol'获得急性脑切片',如'窝烧杯'(图3)和改进的巴斯德吸管滴管(图3和4)中描述的一些材料。有关如何建立这些的完整说明和说明,请参阅Bio-protocol“获取急性脑切片”。
1.巢烧杯
2.改进的巴斯德吸管滴管


图3.巢烧杯


图4.修饰符巴斯德吸管滴管。 打破玻璃一次性巴斯德吸管的最薄端,并将其插入滴管灯泡。

设置:

  1. 准备1 L股ACSF前一天,并存储在4°C过夜(见食谱)。
  2. 在实验当天,准备300毫升的冰冷切片ACSF(见食谱)急性脑切片,并参考Bio-protocol“获取急性脑切片”了解如何获得脑切片的详细程序。

  3. 按照Bio-protocol“获取急性脑切片”的指导,在“巢烧杯”中制备150毫升的恢复ACSF。
  4. 保留其余ACSF(约550毫升)作为“实验ACSF”(见食谱)


  1. 获取大脑切片(或感兴趣组织的外植体)
    见Bio-protocol&nbsp;&nbsp;&nbsp; “获取急性脑片”Papouin和Haydon(2018年)。这是一个非常经典的程序,在许多实验室中用于体外电生理学,各种描述可以在PubMed(例如Ting等人,2014)或Jove网站( https://www.jove.com/video/2330 )。

  2. 孵化切片,以获得条件培养基
    1. 在背侧海马的情况下,通常应从成年雄性小鼠获得6-8个半切片。这可能会根据您的大脑区域或感兴趣的组织而有所不同。一旦恢复结束(Papouin和Haydon,2018年),在装满室温氧合恢复ACSF的陪替氏培养皿(来自巢烧杯,几毫升即可)中分别转移半切片,并仔细分离海马用弹簧剪刀和细镊子切片(图5A)。为了尽量减少对切片的操作和损伤,可以在海马体(马达和躯体感觉皮层通常)上方紧贴一小部分皮层(因此它将参与下面的孵育和条件培养基制备)。将“孤立”的海马切片转移回巢烧杯,并允许额外15分钟的恢复。这种海马分离程序可以通过从培养皿中取出氧合管而得到极大的促进,条件是程序仅需要1或2分钟,切片立即转移回巢烧杯。

      “”src
      图5.孵育海马切片以获得条件培养基A.在含有恢复ACSF的培养皿中,用弹簧剪刀和细钳将海马与切片的其余部分分开。 B.将片段简单转移到实验ACSF中后,用RT实验ACSF将它们转移到装满1.5ml的1.7ml微管中。
      轻轻地氧化,并留在管架上90分钟。

    2. 使用改良的巴斯德吸管(图4),轻轻转移所有的切片在含有氧化实验ACSF的小培养皿(见食谱)在RT。确保切片由于氧合而不旋转。这一步只需要将切片从恢复ACSF转移到调节ACSF(步骤B3)。
    3. 几秒钟后,用改良的巴斯德吸管转移至1.7ml微管底部的3至4个海马切片,并用RT实验ACSF填充至1.5ml。插入充氧管并轻轻充氧(图5B)。降低氧合率,使管内物质不飞溅,切片不旋转。切片应保持在管的底部,轻轻挥手。在管架上在室温孵育90分钟,同时定期检查氧合是否合适。
    4. 小心地用1毫升吸管转移条件培养基(厘米)到一个干净的微管,并立即在-80°C冻结,直到用于D-丝氨酸测量。如果在同一天使用,在4°C储存。注意不要用CM吸移任何组织。在管中保留几微升是更安全的。除非他们区别对待,否则将所有切片孵化的CM集中在一个15毫升的试管中。其余的实验ACSF(即,车辆ACSF)应与CM样品一起储存在-80°C或4°C。在-80°C冷冻保留在管底部的培养组织,直到用Pierce BCA蛋白质测定试剂盒(本方案中未描述)进行蛋白质提取和光密度测量。

  3. 准备生物传感器
    该协议是为了使用Sarissa探头而设计的。它可以很容易地适用于其他D-丝氨酸传感器。如果使用Sarissa D-丝氨酸探针,请根据制造商的说明,准备生物传感器 Sarissa Biomedical (另见Dale等人,2005)或如下:
    1. 通过电生理室运行ACSF(ACSF,用于从海马切片中获得CM),并将室加热器转到33°C。
    2. 一旦流量稳定并达到所需温度,将D-丝氨酸和空传感器探针安装在附着在显微操作器上的生物传感器支架上(图1)。
    3. 使用显微操作器,将探头浸入记录室中,并将它们放置在相互接近并相同的深度(图1和图6)。优选地,两个探针应该以相对于流动相似的角度彼此面对。让传感器重新水化15-30分钟。请注意,在再水合时,传感器会膨胀(像海绵一样)。给传感器足够的空间来补充水分,而不会接触到彼此或接触室的底部。生物传感器的探针部分是非常脆弱的:确保它不会接触任何可能会损坏它的东西。


      图6.生物传感器探头浸入和定位在记录室
      注:在开始运行“D-生物传感器条件培养基”之前,确保灌注线尽可能短。确保灌注率不超过0.5毫升/分钟。确保探头的传感器部分不断被浸没。一旦探针再水化,它们不能暴露在空气中超过几秒钟。将传感器暴露在空气中,同时极化为+500 mV将导致对传感器的直接和不可逆转的损坏。我们强烈建议使用虚拟传感器进行试运行。

    4. 使用软件接口上的双稳压器和极性命令,手动极化+ 500 mV的传感器,并将极性切换到-500 mV并返回,循环约10到12次(见图7)。
    5. 允许偏振至+500 mV至少60分钟,或直到15-20分钟运行过程中电容衰减不可察觉(图7)。


      图7.使用DY2000软件在D丝氨酸传感器(顶部红色迹线)和空白传感器(底部蓝色迹线)上运行的校准实验(10μMD-丝氨酸)的打印屏幕。注意使用的参数,特别是采样率:50Hz,运行持续时间:1,000秒,传感器电位:0.5V和A / V比例因子。请注意,在每次运行结束时,软件都会显示“Ready ...”信号(右下角),表示可以开始新的运行(按RUN按钮,右下角)。但是,刚刚结束的运行不会自动保存。确保在开始另一次运行之前按下保存(左下角,绿色按钮),否则数据将丢失。另外请注意,“噪音”伪影通常出现在两条轨迹上。然而,D-丝氨酸灌注在空传感器上不引起电流。事实上,在无效传感器轨迹上记录1,000秒的过程中,仍然可以检测到约20 pA的电容衰减。

  4. 在生物传感器上运行条件培养基
    1. 在冰上融化条件培养基,然后在室温下离开。管可能包含一些存款。不要通过生产线灌注沉积物,而是将CM移入干净的管中。快速将灌注线从车辆解决方案切换到包含CM的管道。确保线路中没有气泡(我们建议在切换溶液时“挤压”灌注线)。
    2. 一旦CM的体积通过,将生产线切换回车辆解决方案所需的时间,以获得完整的洗入/洗出曲线(图8),并在下一步之前清洗剩余的D-丝氨酸线测量。
    3. 如果在不同的日子使用不同的车辆ACSF获得下一个CM上运行的CM,则运行相应的车辆解决方案至少15分钟,然后运行下一个CM。确保传感器在此过程中不会暴露在空气中。
      注意:运行结束后,DY2000提供的biopotentiostat软件需要保存每次运行的结果。因此,记住在每次运行结束时单击 (图7)是至关重要的。
    4. 看来,不同的传感器倾向于给予非常不同的阅读质量和稳定性。我们建议只进行一次程序D,确定该组生物传感器提供稳定和清洁的安培信号,没有由灌注流或电噪声引起的伪影和波动。在我们的研究中,我们选择中止或排除所有传感器的基线行为将阻止对CM中D-丝氨酸水平的准确可靠评估的所有实验。


      图8. CM D-丝氨酸测量的Clampfit分析的打印屏幕。顶部痕迹:D-丝氨酸传感器。中间痕迹:空传感器。底部痕迹:D-丝氨酸传感器减去空传感器。游标1-2显示在哪里进行基线减法,以便跟踪的基线部分读取0 pA。游标3-4显示测量平均峰值响应的位置。

  5. 校准生物传感器
    1. 预先用载体ACSF中的连续稀释液制备例如0.05,0.5,5和50μM的D-丝氨酸的标准溶液。我们建议提高校准精度,以期望在CM中检测到D-丝氨酸的浓度。
    2. 从最低浓度标准液开始,从载体溶液切换到D-丝氨酸标准溶液。
    3. 通过线路运行每个D-丝氨酸标准溶液,直到达到平台(图9),然后再切换回车辆ACSF。每次使用后,在每次使用溶液之间留出足够的时间彻底洗净(根据您的设置,最高浓度可能需要20分钟)。
      注意:对于程序D和E,当从一种溶液切换到另一种溶液时,确保灌注流不发生中断,以确保室中的液体(和液体流量)保持恒定。否则,可能会危及传感器的浸入,并对其造成不可逆转的损坏。


      图9. 1μMD-丝氨酸校准实验的Clampfit分析的打印屏幕。顶部痕迹:D-丝氨酸传感器。中间痕迹:空传感器。底部痕迹:D-丝氨酸传感器减去空传感器。游标1-2显示基线相减的位置,以便基线跟踪读取0 pA。游标3-4显示了测量平均峰值响应的位置。在1μMD-丝氨酸溶液的灌注开始周围以及〜3ml灌注后有意地产生伪影。这种类型的实验被用来确定测量CM中D-丝氨酸浓度所需的最小液体量。

  6. 丢弃或储存生物传感器
    1. 确保在处理和拔下生物传感器之前,生物传感器不再被极化,并且双稳态器关闭。
    2. 如果要重新使用传感器,请按照制造商的说明进行操作。将传感器快速转移到4°C的补液罐中的缓冲液A(参见制造商的说明书)并置于冰箱中。另外,根据我们的经验,我们发现如果处理得当,传感器可以在冷ACSF(4°C)内保持过夜,并在接下来的48小时内至少重复使用一次,而不会有严重的灵敏度损失。

数据分析

数据分析可以使用Clampfit进行,就像任何电生理追踪(见图8和9)。但是,这需要事先将数据导出为Excel文件。按照这些简单的步骤来做到这一点。

  1. 右键单击要分析的文件。
  2. 用Excel打开。
  3. 删除包含实验细节的第一行。
  4. 可选:如果您知道采样率,请删除时间列。添加一个额外的列,对应于从D-serine传感器(D-ser-Null传感器)的信号中减去空传感器的信号。请注意,这种减法也可以在Clampfit上完成。
  5. 保存为单独的文本文件(.txt)。
  6. 用Clampfit打开那个文本文件。
    注:Clampfit将询问信号的数量,采样率,单位,比例和时间范围。请提供有关上述第4点的相关信息。
  7. 要获得D-丝氨酸 - 空的传感器曲线,如果Excel文本文件没有这样做,请单独保存每条曲线(作为D-丝氨酸和空曲线),并且在两个文件都打开的情况下,只需打开分析&gt;减去控制。 D-serine-Null传感器轨迹将作为单独的单独轨迹出现,并可以另存为一个单独的文件。
  8. 使用Clampfit光标和工具,将基线处的迹线归零并测量D-丝氨酸 - 空值传感器迹线的峰值响应。我们建议在平稳响应的一个或几个分钟内测量平均电流值(以pA为单位)以获得更高的准确度,而不是简单地测量“绝对峰值”,如果迹线通常有噪声,可能会给出错误值(图8和9) 。
  9. D-丝氨酸生物传感器具有100nM的特定下限检测限。但是,在我们的手中,他们成功检测到浓度低至50 nM(运行D-丝氨酸标准时)。实验者可以自行决定是否排除低于指定检测限的D-丝氨酸测量值,或者是否应该将其视为准确的。在我们的研究中(Papouin et al。,2017b),我们选择保守并显示这样的数据点,但将其排除在整体分析之外。

笔记

  1. 用切片孵育的CM的体积(在这种情况下为3ml)应该通过旨在确定可以通过允许生物传感器进行平台检测的灌注系统运行的CM的最小体积的初步实验来设定(参见图9和图例) 。
  2. 尽管空传感器上通常没有观察到电流,但是必须从在D-丝氨酸传感器电极上测量的电流中减去在该传感器电极上测得的电流以获得纯的“D-丝氨酸诱导的”电流分析信号;或“净D-丝氨酸信号”。
  3. 传感器应在每个实验结束时进行校准,以测量其灵敏度。对于前几个实验,我们建议在运行CM之前校准传感器。这将使没有经验的实验者熟悉这个程序。这也表明,如果在实验过程中出现任何问题(如灌注问题和对生物传感器的损害),则仍然可以确定所有CM运行中的D-丝氨酸的绝对浓度,直到然后。然而,不知道是否一次运行几分钟高浓度的D-丝氨酸(50-100μM)导致DAAO活性或完整性的损失,并且是否导致传感器的灵敏度损失。由于CM中的D-丝氨酸水平通常较低,这可能导致在下面的D-丝氨酸测量中缺乏检测或低估。其中一个还存在线路或腔室中存在残留的亚阈值量的D-丝氨酸和随后的CM灌注过程中的错误检测的风险。
  4. 这些生物传感器的经典使用通常包括用生物传感器电极自身穿透大脑切片(Shigetomi et al。,2013)。我们强烈建议不要使用这种方法,因为它将不可避免地导致几十mM的D-丝氨酸的立即峰检测,由于组织破坏和对细胞的不可逆转的损伤,导致细胞内D-丝氨酸(其占脑的99.9%D - 丝氨酸)被释放到细胞外空间。在数十分钟的衰变之后,预计这将稳定到稳态水平,以反映切片中D-丝氨酸的内源性细胞外浓度。相反,这更可能反映出逸出生物传感器周围的瘢痕/濒死组织的残留D-丝氨酸。另外,这种方法高度依赖于传感器被迫进入切片的深度,这决定了传感器的多少与受损组织直接接触,并因此检测到多少D-丝氨酸。最后但并非最不重要的是,我们发现,这种方法可能会导致传感器本身在穿透切片时变差。这些担忧一起促使我们采用本议定书中所述的不同方法。

食谱

  1. 股票人造脑脊液(ACSF)的解决方案(1升,存储在4°C)
    葡萄糖10毫克(1.8克,1升)
    氯化钠125毫克(7克,1升)
    无水磷酸二氢钠1.25mM(1L,0.119g)
    氯化钾2.5mM(1L为0.23mg)
    碳酸氢钠26毫克(2.18毫克,1升)
    调节pH 7.3和渗透压290-300 mOsm L -1
    用ddH <2:O>补足1L
  2. 冰冷切片ACSF(约300毫升)
    股票ACSF
    2 mM氯化镁
    1 mM氯化钙
  3. 恢复ACSF(〜150毫升)
    股票ACSF
    1.5 mM氯化镁
    2 mM氯化钙
  4. 实验ACSF(约550毫升)
    股票ACSF
    1.3 mM氯化镁
    2 mM氯化钙
    注意:其中约3ml将用于获得海马切片条件培养基(调理ACSF ),其余将在安培D-丝氨酸测量期间用作“空白” 车辆ACSF )。标签并在RT处存储以用于&lt; 2天或在-20℃冷冻。每种条件培养基都必须有相应的ACSF。

致谢

这项工作得到了两项菲利普基金会的资助以及授予T.P.的人类前沿科学计划长期奖学金(LT000010 / 2013)和两项授予P.G.H.的NIH / NINDS R01奖学金(NS037585和AA020183)的支持。谁也是GliaCure的创始人。作者声明不存在利益冲突。我们感谢Jaclyn M. Dunphy对本协议的仔细校对。

参考

  1. Dale,N.,Hatz,S.,Tian,F.和Llaudet,E。(2005)。 倾听大脑:用于神经化学物质的微电极生物传感器趋势生物技术< 23(8):420-428。
  2. Ferreira,JS,Papouin,T.,Ladepeche,L.,Yao,A.,Langlais,VC,Bouchet,D.,Dulong,J.,Mothet,JP,Sacchi,S.,Pollegioni,L.,Paoletti,P Oliet,SHR和Groc,L。(2017)。 共激动剂在海马突触处差异性地调节GluN2B-NMDA受体运输 Elife 6。
  3. Johnson,J.W。和Ascher,P。(1987)。 甘氨酸可加强培养的小鼠脑神经元中的NMDA应答 325(6104):529-531。
  4. Kleckner,N.W。和Dingledine,R。(1988)。 甘氨酸激活非洲爪蟾卵母细胞中表达的NMDA受体的要求 科学 241(4867):835-837。
  5. Montesinos Guevara,C.和Mani,A. R.(2016年)。 D-丝氨酸在外周组织中的作用 Eur J Pharmacol 780:216-223。
  6. Papouin,T.,Dunphy,J.M.,Tolman,M.,Dineley,K.T。和Haydon,P.G.(2017a)。 中隔胆碱能神经调节调节海马NMDA受体的星形胶质细胞依赖性门控以调整清醒状态 < Neuron 94(4):840-854 e847。
  7. Papouin,T和Haydon,P.G。(2018)。 获得急性大脑切片。 Bio-protocol 8(2):e2699 。
  8. Papouin,T.,Henneberger,C.,Rusakov,D.A。和Oliet,S.H.R。(2017b)。 星形胶质细胞与神经元D-丝氨酸:事实检查 趋势Neurosci < 40(9):517-520。
  9. Papouin,T.,Ladepeche,L.,Ruel,J.,Sacchi,S.,Labasque,M.,Hanini,M.,Groc,L.,Pollegioni,L.,Mothet,JP和Oliet,SH(2012) 。 突触和突触外NMDA受体由不同的内源性参与者门控 / 150(3):633-646。
  10. Pernot,P.,Mothet,J.P.,Schuvailo,O.,Soldatkin,A.,Pollegioni,L.,Pilone,M.,Adeline,M.T.,Cespuglio,R.and Marinesco,S。(2008)。 在中枢神经系统中检测D-丝氨酸的酵母D-氨基酸氧化酶微生物传感器的特征。 Anal Chem 80(5):1589-1597。
  11. Shigetomi,E.,Jackson-Weaver,O.,Huckstepp,R.T.,O'Dell,T.J.,Khakh,B.S. (2013年)。 J Ne urosci 33(24):10143-53。
  12. Ting,J.T.,Daigle,T.L.,Chen,Q.,Feng,G。(2014)。 成年动物和衰老动物的急性脑切片方法:应用靶向膜片钳分析和光遗传学。 a> Methods Mol Biol 1183:221-42。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Papouin, T. and Haydon, P. G. (2018). D-serine Measurements in Brain Slices or Other Tissue Explants. Bio-protocol 8(2): e2698. DOI: 10.21769/BioProtoc.2698.
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