参见作者原研究论文

本实验方案简略版
Mar 2018

本文章节


 

Preparing Viable Hippocampal Slices from Adult Mice for the Study of Sharp Wave-ripples
成年小鼠海马脑片的制备及其锐波波纹的研究   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

We describe a protocol for preparing acute brain slices which can produce robust hippocampal sharp wave-ripples (SWRs) in vitro. The protocol is optimized for its simplicity and reliability for the preparation of solutions, slicing, and recovery incubation. Most slices in almost every mouse prepared though the protocol expressed vigorous spontaneous SWRs for ~24 h, compared to the 20-30% viability from "standard" low sodium slicing protocols. SWRs are spontaneous neuronal activity in the hippocampus and are essential for consolidation of episodic memory. Brain slices reliably expressing SWRs are useful for studying memory impairment and brain degeneration diseases in ex vivo experiments. Spontaneous expression of SWRs is sensitive to conditions of slicing and perfusion/oxygenation during recording. The amplitude and abundance of SWRs are often used as a biomarker for viable slices. Key improvements include fast circulation, a long recovery period (3-6 h) after slicing, and allowing tissue to recover at 32 °C in a well perfused incubation chamber. Slices in our custom-made apparatus can express spontaneous SWRs for many hours, suggesting a long period with balanced excitation and inhibition in the local networks. Slices from older mice (~postnatal 180 days) show similar viability to younger (postnatal 21-30) mice.

Keywords: Brain slice (脑片), Hippocampus (海马体), Sharp wave/ripples (锐波波纹), Electrophysiology (电生理学), Oscillations (振动), Brain tissue viability (脑组织活力)

Background

Acute brain slices have been an invaluable preparation for electrophysiology and other neuroscience studies. While there is a vast body of literature describing the usage of brain slices, protocols for reliably producing viable slices are usually discussed in less detail. Slices from older animals are typically less viable. Viability of the brain slices is often described using healthy cell morphology and electrophysiological properties of the neurons. However, healthy looking neurons in the tissue do not always translate to normal network behaviors. For example, thin slices prepared with the standard sucrose replacement protocol (e.g., Aghajanian and Rasmussen, 1989; Ye et al., 2006) provide morphologically healthy cells for patch studies, but do not express spontaneous network activity such as hippocampal sharp-wave ripples (SWRs).

SWRs are spontaneous neuronal population events that occur in the hippocampus during sleep and quiet restfulness (for review see Buzsáki, 2015). An experience, such as an exploration of the environment, is registered in the hippocampus as sequential activations of neuronal assemblies (a.k.a “place cell assemblies”). These sequences are then re-activated in SWRs during sleep or quiet restfulness (off-line states), and contribute to the consolidation of the memory (Wilson and McNaughton, 1994; Lee and Wilson, 2002; Ji and Wilson, 2007). Suppression of SWRs impairs hippocampus dependent episodic memory (Ego-Stengel and Wilson, 2010), suggesting that SWRs are important for the reactivation of experience related to neuronal ensembles in the absence of related sensory input.

Spontaneous SWRs also occur in vitro in hippocampal slices (Kubota et al., 2003; Maier et al., 2003 and 2009; Colgin et al., 2004; Behrens et al., 2005; Miyawaki et al., 2014; Keller et al., 2015). However, in standard submerged-type recording chambers, SWRs are rarely seen, due to insufficient perfusion. Vigorous perfusion on both sides of the slice seems to be an essential condition for providing adequate oxygen that allows for the SWRs to occur (Wu et al., 2005; Hájos et al., 2009; Maier et al., 2009).

In our experience, slices prepared with standard high-sucrose low-sodium protocol (Aghajanian and Rasmussen, 1989) only have a lower probability (20-50%) of expressing spontaneous SWRs. Slices from mice older than postnatal (P) 30-days have a further reduced probability of expressing SWRs and often fail to induce LTP. Extending the recovery time after slicing often helped. Consistent with (Maier et al., 2009), we also observed that SWRs were rarely expressed in conventional slice incubation chambers, which have passive fluid convection or oxygen bubble induced convection (e.g., Brain Slice Keeper-4 apparatus, Automate Scientific). A forced fluid circulation with a small pump significantly increased SWR expression. We have tested 198 slices from 35 mice of P 35 to P180 days of age, and every animal tested showed spontaneous SWRs, including 6-month-old 5xFAD mice with obvious amyloid plaques and slices from mice that have undergone severe traumatic brain injuries.

Fast circulation may be important for two reasons: 1. Thick slices (350-500 μm) are essential for preserving SWR generating circuits. Fast circulation on both sides of the slice can improve the oxygen delivery into the thickness of the tissue. 2. SWRs are spontaneous activity occurring 1-2 times/second. Continuous activation of large number of neurons requires more oxygen provided by fast circulation.

Our protocol is also aimed at its simplicity, i.e., using stock solutions to minimize the solution preparation time (< 30 min), using simple low sodium high sucrose cutting solution without unstable compounds like NMDG (Tanaka et al., 2008) and antioxidants (Brahma et al., 2000), and omitting the complex procedure of intra-cardiac perfusion and protective recovery protocol (Ting et al., 2014 and 2018). Some of our research papers using the protocol have been published (Jiang et al., 2018; Sun et al., 2018; Li et al., 2019).

Materials and Reagents

  1. Paper towel
  2. Glass pipet for local field potential electrode, 1.5 mm outer diameter x 1.1 mm inner diameter, 7.5 cm long borosilicate glass pipet with inner filament (Sutter Instruments, catalog number: BF150-110-7.5 )
  3. Nylon mesh (Component supply UMN-600), glued onto the bottom of the slice holder
  4. Silicon tubing (1/4-3/8, VWR , catalog number: 89403-862), for connecting the pump and the top chamber
  5. Disposable Transfer Pipettes (Thermo Scientific, Samco 3.2 ml) for slice transferring between incubation and recording chamber
  6. Animals
    Wildtype Mice (C57BL/6J, RRID: IMSR_JAX :000664, Jackson Labs) and transgenic 5xFAD mice (RRID:MMRRC_034840-JAX, Jackson Labs) (Oakley et al., 2006) were used. Mice for experiments were from our Georgetown colony, they were kept on a standard 12 h light/dark cycle, food and water were provided ad libitum, with all experimental procedures performed in accordance with the guidelines of the Georgetown University Animal Care and Use Committee.
  7. Super glue (liquid form, Loctite, catalog number: 1775049) for gluing the brain during slicing
  8. Sucrose (ACS Reagents, Fisher Scientific, catalog number: S5-500 )
  9. KCl (ACS Reagents, Fisher Scientific, CAS: 7447-40-7 )
  10. MgSO4 (ACS Reagents, Fisher Scientific, CAS: 7487-88-9 )
  11. CaCl2 (ACS Reagents, Fisher Scientific, catalog number: C79-500 )
  12. Glucose (ACS Reagents, Sigma-Aldrich, catalog number: G8270-5KG )
  13. NaH2PO4 (ACS Reagents, Sigma-Aldrich, catalog number: S0751-1KG )
  14. HEPES (ACS Reagents, Fisher Scientific, catalog number: BP310-1 )
  15. NaCl (ACS Reagents, Sigma-Aldrich, catalog number: S5886-10KG )
  16. Carbogen (95% O2, 5% CO2, tank and regulator available from local gas suppliers)
  17. NaHCO3 powder (ACS Reagents, Fisher Scientific, catalog number: S233-500 )
  18. Slice cutting ACSF solution (see Recipes)
    Stock 1: Sucrose
    Stock 2: Cutting ACSF
    Stock 3: NaHCO3 powder
  19. Incubation/recording ACSF (see Recipes)

Equipment

For slice preparation

  1. Vibratome slice cutter (Leica, model: VT1000S )
  2. Water bath (e.g., Southwest Science , model: SHW10LD 10 Liter Bath)
  3. Digital Thermometer ( VWR Traceable Double Thermometer, catalog number: 23226-656 , or any type with 0.1 °C accuracy)
  4. Custom-made incubation chamber illustrated in Figure 2, made of 1/4 Plexiglass (acrylic) sheet, obtained from McMaster-Carr
  5. Slice holder is cut from an egg crate light diffusor panel with 1/2 x 1/2 x 1/2 cells (e-Plastics SKU: W/EGG.500X2X4)
  6. 600 ml glass beaker ( VWR , catalog number: 89001-078 )
  7. Carbogen blower (e.g., Air Stone Cylinder Bubble Diffuser from Amazon) three of them are needed (two for incubation gas blowers for incubation chamber and recording ASCF flask, one used as a filter for ACSF circulation in recording chamber)
  8. Low voltage, low flow rate pump, available from a solar powered mini water fountain/bird bath kits (Amazon). The pump provides a flow of ~1,000 ml/min, and uses a safe low voltage power supply (below)
  9. Variable DC power supply for the pump (Circuit specialist CSI5003XE, or B&K Precision 1550 DC Power Supply)
  10. Surgical scissors for mouse decapitation (WPI, catalog number: 501743G )
  11. Spring scissors (WPI, catalog number: 15905G ) for cutting through skull and extracting the brain
  12. Size 4 watercolor pointed brush (Office depot 596713) for transferring brain slices
  13. Dumont #7 student forceps (Fine Science Tools, catalog number: 97797-00 ) for extracting the brain
  14. Tapered Spatula (PTFE coated, VWR , catalog number: 10806-412) for extracting the brain
  15. Dumont #5 student forceps (Fine Science Tools, catalog number: 91150-20 ) for adjusting slice position in recording chamber

For SWR recording
While most electrophysiology rigs can be used for recording SWRs, perfusion of both sides of the slice with a high circulation rate is essential. See Maier et al. (2009) and Hájos et al. (2009) for the modification of conventional chambers on patch recording rigs. Listed below are parts for a low-cost local field potential recording system. The system is easy to use and reliable for recording many hours of SWRs from one slice.

  1. Local field potential recording chamber
    A chamber that can perfuse both sides of slice is important. See procedures and data recording sections. Figure 3 illustrates the flow directions in the custom-made recording chamber.
  2. LFP amplifier, 1000x amplification with a probed head stage (e.g., Warner Instruments, model: DP 311A )
  3. Data acquisition system to digitize the data (e.g., NeuronNexue smart box, allowing multiple channels with an electrode array)
  4. An oscilloscope (e.g., Digikey, 2250-SDS1022-ND ) for positioning electrode
  5. An audio monitor (A-M Systems 3300) for positioning the electrode
  6. Manual micromanipulators (SD Instruments SKU MX160 ) and magnetic stands for holding the manipulators (SD Instruments)
  7. 625 nm LED (Thorlabs, model: M625D3 ) for slice illumination under the chamber
  8. Pipet puller (Sutter, model: P97 ) for pulling ultra-low impedance pipet for local field potential (LFP) recordings
  9. LFP recording and Stimulation electrodes (low impedance (~500K) glass pipette pulled by the pipette puller). Low electrode impedance (50-500K) is important for low noise recording.
  10. Peristaltic pump, for ACSF circulation (e.g., Pulsatron Dolphin Series Peristaltic Metering Pump, 60.0 gpd/25 PSI, Norprene tubing, .25" ID x .44" OD).
  11. Plastic tubing for connecting pump and recording chamber perfusion ( VWR , catalog number: 89403-850 , 3/32-5/32 Tygon tubing)
  12. Tubing clamp flow regulator ( VWR Talon® Regular Hosecock Clamp), for regulating perfusion rate of the recording chamber
  13. 1000 ml glass flask for circulating ACSF perfusion of the recording chamber ( VWR , catalog number: 75804-652 )
  14. Stereo zoom dissecting microscope, 10-40x zoom, available from Olympus, Nikon, or American microscopes (SKU: V-SM-3 SD Instruments)
  15. Recording bench (Newport, catalog number: IG-22-2 )
    This 2 x 2 ft optical breadboard can be set up on standard laboratory bench top. Recording the LFP from SWRs does not need a vibration isolation table. A Faraday cage may also be avoided if a low impedance glass electrode is used and there is no instrument power cord within 30-inches of the set up.

Procedure

  1. Preparation
    Before slicing, prepare two artificial cerebral-spinal fluid (ACSF) solutions from the stock solutions (procedures in the recipe section below): The cutting ACSF (200 ml) and incubation ACSF (2,000 ml). Both are vigorously bubbled for ~3 min with carbogen gas. The cutting ACSF is then cooled down to 4 °C in an ice/water bath, and the incubation ACSF is warmed up to 32 °C in the incubation apparatus (see details in the Recipes section).

  2. Slicing
    1. The procedures for using animals are strictly following a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at Georgetown University Medical Center following the NIH guidelines.
    2. The animal is deeply anesthetized in an anesthesia jar (approved by IACUC) by freely breathing isoflurane vapor (> 5%) for ~20 s. After being fully anesthetized (loss of muscle tension and no response to tail pinch), the animal is quickly decapitated with a pair of surgical scissors.
    3. Use a pair of small surgical scissors to cut the skin along the midline of the scalp to expose the skull, then immerse the head in the cold (4 °C) cutting ACSF solution for ~1 min. While immersed, remove the tissue covering the skull to reduce heat insulation. The brain should be cooled down to 5-10 °C after this step.
    4. Use a pair of small surgical scissors to cut the skull following Figure 1. Use the Dumont #7 forceps to remove the skull fragments over the brain. Gently extract the brain with the spatula, starting from the rostral end, and let the brain slide into the cutting ACSF solution at ~4 °C.
      Important: Make sure that the tip of spatula stays at the midline of the ventral side of the brain, to avoid touching the ventral temporal lobes which may lead to compression of the ventral hippocampus. The temperature of the cutting ACSF needs to be higher than 0 °C to avoid damage caused by hypothermia.


      Figure 1. Skull cutting lines for brain extraction. Red lines mark the cuts through the skull. Numbers are the suggested sequence. Cut 5 should be not too deep to ensure no disturbances to the cortical tissue beneath the cut. The head needs to be cooled down to below 10 °C before brain extraction. As long as the brain is cold, it is not critically important for a short extraction time. Attention should be paid not to stretch or compress the hippocampi during extraction.

    5. Apply a thin layer of super glue liquid on the surface of the vibratome cutting stage for gluing the brain on the stage.
    6. Use the spatula to pick up the brain from the cutting solution (dorsal side of the brain up), and use a small piece of paper towel to absorb excess solution from the ventral side, and then place the ventral side of the brain onto the surface of the cutting plate coated with the super glue liquid.
    7. Apply a small amount of pressure onto the dorsal side of the brain to secure a firm gluing contact between the brain and the cutting stage.
    8. Now mount the cutting stage onto the vibratome, and fill the chamber of the cutting stage with 4 °C cutting ACSF. The brain is sliced in a horizontal orientation, from the rostral end to the caudal end. A 12.7-degree tilting angle from the horizontal plain (high at the rostral end) may better preserve the connection between CA3 and CA1 areas for better SWR expression (Miyawaki et al., 2014). However, when cut at 480-500 μm, the 12.7 degree of tilting angle is not important. Thicker slices (> 350 μm, Maier et al., 2009) are very important for the expression of SWRs.
    9. Hippocampal slices (that we cut at 488 μm) are cut at a high vibrating speed (~70 Hz) and a low preceding speed (1-2 on the vibratome setting). It is OK to allow the temperature of the cutting ACSF solution to elevate from ~4 °C to 10 °C while cutting. Bubbling with carbogen is needed if the total cutting time is longer than 30 min, to prevent a change in the pH of the cutting solution.
    10. After each cut, the slice is gently transferred to the incubation ACSF in the incubation chamber using the fine paint brush while paying attention not to bend or compress the tissue. The whole horizontal slice (two hemispheres) can be trimmed into two halves before transferring. Usually we obtain 4-5 whole horizontal slices from each animal (10 hippocampal slices from both sides).

  3. Incubation
    1. Incubation is maintained at 32 °C in the incubation apparatus (Figure 2), with constant flow circulation. Adjust the variable power supply and maintain a rate of ~1,000 ml/min (3.5-4 V to the pump). The flow rate should not be higher than 1,000 ml/min, in order to reduce movement or floating of the slices in the solution which may cause mechanical damage to the tissue.
    2. Carbogen should be constantly bubbled into the ACSF in the beaker at a rate of 200-400 ml/min, to maintain saturation of O2/CO2 in the ACSF solution.
    3. After 2 h at 32 °C, reduce the water bath temperature to 27 °C and allow the ACSF to slowly cool down from 32 °C to 27 °C (it takes about 1.5 h). Continue incubating until the slices are used. Slices stay viable for ~24 h in the incubation apparatus.


      Figure 2. Construction of the incubation apparatus. The upper container of the apparatus is a container of flowing ACSF, made of acrylic plastic glass. Inside the container is a slice holding chamber (cut from an egg crate style lamp diffuser [see Equipment]). Slices are held in separated compartments (top view), and immersed in circulating ACSF solution. Nylon mesh (see Materials) is glued to the slice holder to allow good circulation. The bottom side of the slice holder has a higher flow rate than that of the top side, that provides a small downward current in each compartment to stabilize the slices against the mesh. A barrier in the top container maintains the fluid level. Below the apparatus is a 600 ml glass beaker, sitting in a water bath with water ~6 cm deep to maintain the temperature of 32 °C. ACSF in the beaker is continuously bubbled with carbogen at a rate of 200-400 ml/min. A low voltage pump (see Equipment) is used for circulating the ACSF. The pump is powered by a variable DC power supply (see Equipment). Adjust the voltage to the pump (3-4 V) to have a flow of rate of ~1,000 ml/min (about 1/2 of the pump capacity). The total fluid volume is 800-1,000 ml. The upper container is covered to reduce evaporation and a change in osmolarity after many hours.

  4. SWR recording
    1. Establish a circulation of the incubation/recording ACSF solution in the recording chamber: Put 800-1,000 ml of incubation ACSF into the 1,000 ml flask, put the flask on a shelf 1-1.5 meters above the level of the recording chamber to allow a gravitational flow and syphon it from the flask to the chamber via the 3/32x 5/32 Tygon tubing. An air stone is connected to the tubing end in the flask and serves as a fluid filter for the circulation and as an anchor for the tubing to stay at the bottom of the flask. Suction by laboratory vacuum or a syringe is used to prime the syphon. The flow rate is regulated by the tubing clamp. The normal circulation rate is 40-100 ml/min. The fluid dropped out of the recording chamber is collected in a 100 ml beaker below the chamber and sent back to the flask by a peristaltic pump. We use gravitational force for both inlet and outlet flow of the ACSF as this can completely remove the static electric noise generated by squeezing the tubing while running the peristaltic pump. The ACSF in the flask is continuously bubbled with carbogen at a rate of 50-100 ml/min, to maintain saturation of O2/CO2.
    2. SWRs gradually emerged after > 3 h of incubation. After 7 h of incubating, most of the slices should express SWRs from the dorsal most to the ventral most of the hippocampus.
    3. To position a slice in recording chamber: Carefully transfer a slice from the incubation chamber into the center of the recording chamber by a fluid transfer pipette (the small end of the pipette is cut out). The slice should be held stable at the center of the chamber by the vertical flow of the fluid. The position and orientation of the slice can be gently adjusted by a pair of #5 fine forceps. Pay great attention not to bend or compress the tissue. A mechanical disturbance to the tissue can stop or attenuate SWRs for a few minutes. The hippocampus should be positioned at the center of the view field of the stereo microscope at its full zoom magnification (40x).
    4. To prepare the recording electrode: Ultra-low resistance glass electrode is pulled by 5-7 pulls using the P-97 puller. Multiple pulls result in resistance of 50-100K Ohms when filled with ACSF, and the tip opening is about 10 μm. Low resistance leads to low noise for the LFP recording.
    5. Mount the electrode onto the head stage of the amplifier. Put the tip of the electrode into the fluid surface in the recording chamber over the top of the hippocampal slice. This step can be done without the microscope. Turn on the oscilloscope and the audio monitor. There should be a flat baseline in the oscilloscope and a small static noise in the audio monitor. A jumping baseline and large audio noise indicate the electrode is not filled well (air bubbles). Measure the resistance of the electrode, it should be less than 100K Ohms.
    6. To position the electrode tip: Turn the zoom magnification of the microscope down to the minimum (0.75x) and adjust the focus to find the electrode tip. Adjust the tip's position with the manipulator to lower the tip to approach the recording position (stratum radiatum of CA1, Figure 4A). When adjusting the electrode, always adjust the microscope focus so the electrode tip is in sight. Never move the electrode if its tip is not in the focus of the microscope. At this time, do not insert the tip into the tissue. Now increase the zoom magnification of the microscope to 2-3x, adjust the focus to find the electrode tip. At the high zoom power continue to lower down the electrode until it sits on the surface of the tissue (both the slice surface and the electrode tip is in focus). Now the sound of the ripple oscillation (~100 Hz) should be audible in the audio monitor.
    7. To insert the electrode tip into the tissue: Slowly advance the electrode tip to the surface of the tissue. The ripple sound becomes louder when approaching the tissue. When the tip touches the tissue, a loud noise can be heard in the audio monitor. At the contact position, slowly turn the knob of the insertion axis of the manipulator 1/4-1/2 turns, which advances the tip 90-180 μm into the tissue. Note that the mechanical disturbance of the electrode can attenuate the amplitude of the SWRs for a few minutes, so slow insertion is important (~20 s for 90 μm advance). Such small and slow movement of the electrode tip cannot be seen by the microscope, so guiding by audio monitor is needed. After a few minutes of electrode insertion, vigorous SWRs with high amplitude should appear and be sustained for hours (Figure 4). We usually recorded 100-200 μm below the surface where the signal is the largest.
    8. Slices from dorsal hippocampus have a much slower SWR rate than those from the ventral hippocampus. SWRs will only be sustained when both sides of the slice are well perfused in a submerged recording chamber. SWRs do not express well in conventional single sided perfusion chambers (e.g., Warner RC-22).
    9. The sustaining of SWRs during recording: In our two-sided perfusion chamber shown in Figure 3, SWRs can be sustained for ~24 h (Figure 4C, Figure 5).


      Figure 3. Recording chamber. The schematic drawing illustrates a custom-made recording chamber that creates a downward vertical fluid flow around the slice. The flow provides vigorous perfusion to both sides of the slice. Also, the downward flow keeps the slice stable (no need for a weight on the slice). We use a barrier to maintain the fluid level on top of the tissue. The outlet of the ACSF is gravity dripping, which significantly reduces the electrical noise introduced by vacuum suction. Also, the ACSF can be collected and re-circulated by a peristaltic pump. A light emitting diode (LED) chip on the bottom of the chamber is used to provide diffuse illumination. Using red LED (630 nm) can avoid photoactivation of the neurons expressing channel rhodopsin-2. Red light also increases the transparency of the thick tissue. This chamber is thick and will not fit onto a compound microscope for patching cells. However, SWRs do not express well in conventional single-side perfusion chambers (e.g., Warner RC-22). Ensuring that both sides of the tissue are perfused well is important (Hájos et al., 2009; Maier et al., 2009; Caccavano et al., 2020) for the SWRs to express well in chambers for the cell patch recordings.


      Figure 4. SWRs from young and old mice. A. left: SWRs were recorded from striatum radiatum by an ultra-low resistant glass pipet (< 100K ohms, filled with saline) that provides excellent signal-to-noise ratio. Right traces: Abundant SWRs were seen in young mice with high sharp wave amplitude (top trace), and ripple oscillations (bottom insert, filtered between 60-180 Hz for clarity). B. All older mice tested (P6 month, 4 out of 4) expressed SWRs. The SWRs had lower amplitude and rate compared to the young mice. C. SWRs from a slice of the same animal in B, recorded after overnight perfusion in the holding chamber. While the amplitude and repeating rate were further reduced, the occurrence rate of SWRs didn't show a large decline.

Data analysis

SWRs can be clearly distinguished from seizure/interictal spikes by its occurrence rate, LFP amplitude, and calcium signals (Li et al., 2019). SWRs have a high occurrence rate (1-3 events/s) while interictal spikes are much slower (0.3-0.01 events/s). SWRs are also accompanied by ~100 Hz ripple oscillations (Figure 4A insert), but interictal spikes are mostly accompanied by low gamma (20-50 Hz).
    Recovery in the incubation apparatus is important for SWR expression. In our experience with 198 slices, SWRs were never seen from slices that recovered for less than 2 h, but always seen after 7 h of recovery time. Usually, younger mice (P28-P35) recover sooner, and 3-4 h of incubation is adequate. Older mice (P60-P180) take a longer time to recover from slicing, but after 5-7 h, most of the slices show SWRs. In animals genetically encoded with GCaMP-6f, hippocampal regions show strong green fluorescence after cutting, which suggests an elevated concentration of intracellular calcium. The fluorescence gradually reduced after a few hours of recovery in the incubation chamber. In conventional beaker-and-oxygen-blower type incubation chambers, often only a fraction of slices recovered well and expressed SWRs. The fluid circulation in conventional chambers were not well controlled. When oxygen delivery was less optimized, the viability of the tissue declined at the same time as the recovery from the trauma of slicing. Longer recovery time would not improve the viability if the ACSF circulation is compromised.
    To improve the fluid circulation during the recovery period, we have fabricated a number of custom-made incubation chambers for longer recovery time. The common feature for these chambers is a forced circulation either by a stir bar or a pump. We found that ordinary peristaltic pumps do not provide adequate flow for a large incubation chamber which holds ~26 slices from 2-3 animals. Thus, we used a low voltage DC pump (obtained from a solar fountain) with a flow of ~1,000 ml/min. This high flow greatly improved the circulation of ACSF around all the slices. Better oxygenation also allows the slices to recover in a higher temperature (~32 °C). The apparatus brings a stable condition for recovery from cutting related cellular injury and elevated intracellular calcium. Part of our work using thick slices and forced circulation have been published (Sun et al., 2018; Jiang et al., 2018; Li et al., 2019). Slices from older (P3-6 month) animals also have apparent normal SWR expression prepared by this protocol (Figure 4B/C) despite traumatic brain injury (unpublished observations) or the development amyloid plaques in the 5XFAD animals (Caccavano et al., 2020).
    In most slices, the amplitude of the SWRs gradually reduced with time in the recording chamber. It took ~5 h in the recording chamber for the SWR amplitude to reduce to half (Figure 5). It seems that 6-month old mice had more amplitude reduction than those in one-month old animals. It also appears that after long recording, the SWR amplitude was less diverse; more SWRs with similar amplitude were seen (Figure 5).
    It seems that slices with no spontaneous SWRs also showed poor performance in evoked activities, e.g., failed to be potentiated by a tetanus stimulation (100 Hz x 100 pulses) and low amplitude in theta-gamma oscillations when bathed in carbachol (20 mM) containing ACSF. These less viable slices were often subjected to shorter recovery time after cutting (unpublished observations).
The biomarker of slice "viability" is an important issue. In this manuscript, we consider a slice viable if SWRs are vigorously expressed for hours. Expressing SWRs requires network integrity, with balanced synaptic excitation/inhibition (Schlingloff et al., 2014).


Figure 5. Recording time and amplitude declining. A. Continuous recording (>10 h) of SWR amplitude from a slice of P30 animal. The recording started at 10 h after slicing. SWR amplitude gradually reduced over the entire period. The reduction rate was to one-half in 5 h. B. Traces from 10 and 23 h after slicing. Note that the SWRs with lower amplitude displayed a disproportionate reduction after may hours. In this Figure we show SWR signals from striatum oriens (signal polarity is reversed from that in Figure 4).

Recipes

  1. Slice cutting ACSF solution (200 ml per experiment, made before use from two stock solutions and 0.43 g NaHCO3 powder)
    Final composition: Sucrose (252.0 mM); KCl (3.0 mM); MgSO4 (4.0 mM); CaCl2 (1.0 mM); Glucose (10.0 mM); NaHCO3 (10.0 mM); NaH2PO4 (1.25 mM); HEPES (5.0 mM); Saturated with carbogen; pH = 7.4.

    Stock 1: Sucrose
    1. 177 g sucrose, dissolved in 1,000 ml hot (65 °C) distilled water
    2. Then add distilled water to a final volume of 2,000 ml
    3. Keep the stock at 4 °C before use
    The 2,000 ml stock solution is good for 10 experiments (180 ml per experiment), keeping at 4 °C and use within one month.

    Stock 2: Cutting ACSF
    1. Dissolve the chemicals in the following sequence in 180 ml distilled water
      Reagent (Molecular weight)
      Grams for 200 ml
      KCl (74.55)
      0.45 g
      MgSO4 (120.37)
      0.96 g
      Glucose (180.8)
      3.60 g
      NaH2PO4 (119.98)
      0.30 g
      HEPES (238.3)
      2.38 g
      CaCl2·2H2O (147.02)
      0.29 g
    2. After all chemicals are fully dissolved, add distilled water to final value of 200 ml
    3. Keep the stock in 4 °C refrigerator before use
    The 200 ml stock solution is good for 10 experiments (20 ml for each experiment, keep at 4 °C and use within one month).

    Stock 3: NaHCO3 powder
    Prepare 10 of 0.43 g NaHCO3 powder (each store in a 1.5 ml Eppendorf tube) for 10 experiments
    This significantly reduce the preparation time before each experiment

    Making 200 ml slice cutting ACSF solution before each experiment:
    1. Take 180 ml of sucrose stock solution and add 20 ml of cutting stock solution to reach a final volume of 200 ml. Vigorously stir-mix the two solutions with a magnetic stir bar.
    2. Put the NaHCO3 powder in one Eppendorf tube (0.43 g) into the 200 ml slice cutting ACSF. Add the powder slowly while vigorously stirring until all the fine crystals of NaHCO3 are dissolved. Blow carbogen with a fish tank air stone for 3 min to ensure full saturation of O2/CO2. Cool the solution down to 4 °C before use.
      Important: Measure the temperature of the cutting solution before use. The optimum temperature for slicing is 3-6 °C. Because both stock solutions were at 4 °C before mixing, it should take only about 10 min to cool down the cutting ACSF solution.
    3. Incubation ACSF (2,000 ml per each experiment, made from the stock solution below and 4.37 g of NaHCO3 powder).
    Final Composition of incubation ACSF: NaCl (132.0 mM); KCl (3.0 mM); MgSO4 (2.0 mM); CaCl2 (2.0 mM); Glucose (10.0 mM); NaCO3 (10.0 mM); NaH2PO4 (1.25 mM); HEPES (5.0 mM). Saturated with carbogen, pH =7.4, Osmolarity 305-310

  2. Incubation/recording ACSF
    Stock solution for incubation/recording ACSF
    1. Dissolve the chemicals in the following sequence in 1,600 ml distilled water
      Reagent (Formula weight)
      Grams for 2,000 ml
      NaCl (58.44)
      144.0 g
      KCl (74.55)
      4.5 g
      MgSO4 (120.37)
      4.8 g
      Glucose (180.8)
      36.0 g
      NaH2PO4 (119.98)
      3.0 g
      HEPES (238.3)
      23.8 g
      CaCl2·H2O (147.02)
      5.9 g
    2. After all chemicals are fully dissolved, add distilled water to reach a final volume of 2,000 ml
    3. Keep the stock solution at 4 °C and use within one month. The 2,000 ml ACSF stock solution is good for 10 experiments
    4. Prepare 20 of 4.37 g NaHCO3 powder (each store in a 17 ml capped culture tube) for 20 experiments. This significantly reduce the preparation time before each experiment

    Making 2,000 ml incubation ACSF before each experiment:
    1. Take 200 ml incubation ACSF stock and dissolve in 1,600 ml warm (35 °C) distilled water
    2. Stir-mix the solution with a magnetic stir bar
    3. Add NaHCO3 powder from one tube (4.37 g) into the solution while vigorously stirred until fine crystals of NaHCO3 are fully dissolved
    4. Add warm (35 °C) distilled water to a reach a final volume of 2,000 ml
    5. Bubble with carbogen for 3 min to ensure fully saturated with O2/CO2
    Note: Measure the temperature of the ACSF, it should be ~32 °C before filling into the incubation apparatus.

Acknowledgments

Supported by NIH R03AG061645, Georgetown University Medical Center Dean's Toulmin grant FY2017, FY 2019.

Competing interests

The authors declare no competing financial interests.

Ethics

The procedures for using animals are strictly following a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at Georgetown University Medical Center following the NIH guidelines.

References

  1. Aghajanian, G. K. and Rasmussen, K. (1989). Intracellular studies in the facial nucleus illustrating a simple new method for obtaining viable motoneurons in adult rat brain slices. Synapse 3(4):331-338.
  2. Behrens, C. J., van den Boom, L. P., de Hoz, L., Friedman, A. and Heinemann, U. (2005). Induction of sharp wave-ripple complexes in vitro and reorganization of hippocampal networks. Nat Neurosci 8(11): 1560-1567.
  3. Brahma, B., Forman, R. E., Stewart, E. E., Nicholson, C. and Rice, M.E. (2000). Ascorbate inhibits edema in brain slices. J Neurochem 74:1263-1270.
  4. Buzsáki, G. (2015). Hippocampal sharp wave-ripple: A cognitive biomarker for episodic memory and planning. Hippocampus 25(10): 1073-1188. 
  5. Caccavano, A., Bozzelli, P. L., Forcelli, P. A., Pak, D. T. S., Wu, J. Y., Conant, K. and Vicini, S. (2020). Inhibitory parvalbumin basket cell activity is selectively reduced during hippocampal sharp wave ripples in a mouse model of familial Alzheimer's disease. J Neurosci doi: 10.1523/JNEUROSCI.0425-20.2020.
  6. Colgin, L. L., Kubota, D., Jia, Y., Rex, C. S. and Lynch, G. (2004). Long-term potentiation is impaired in rat hippocampal slices that produce spontaneous sharp waves. J Physiol 558(Pt 3): 953-961.
  7. Ego-Stengel, V. and Wilson, M.A. (2010). Disruption of ripple-associated hippocampal activity during rest impairs spatial learning in the rat. Hippocampus 20(1):1-10.
  8. Hájos, N., Ellender, T. J., Zemankovics, R., Mann, E. O., Exley, R., Cragg, S. J., Freund, T. F. and Paulsen, O. (2009). Maintaining network activity in submerged hippocampal slices: importance of oxygen supply. Eur J Neurosci 29(2): 319-327.
  9. Jiang, H., Liu, S., Geng, X., Caccavano, A., Conant, K., Vicini, S. and Wu, J. (2018). Pacing hippocampal sharp-wave ripples with weak electric stimulation. Front Neurosci 12: 164.
  10. Ji, D. and Wilson, M. A. (2007). Coordinated memory replay in the visual cortex and hippocampus during sleep. Nat Neurosci 10(1): 100-107.
  11. Keller, M. K., Draguhn, A., Both, M. and Reichinnek, S. (2015). Activity-dependent plasticity of mouse hippocampal assemblies in vitro. Front Neural Circuits 9: 21.
  12. Kubota, D., Colgin, L. L., Casale, M., Brucher, F. A. and Lynch, G. (2003). Endogenous waves in hippocampal slices. J Neurophysiol 89(1): 81-89.
  13. Lee, A. K. and Wilson, M. A. (2002). Memory of sequential experience in the hippocampus during slow wave sleep. Neuron 36(6): 1183-1194.
  14. Li, P., Geng, X., Jiang, H., Caccavano, A., Vicini, S. and Wu, J. Y. (2019). Measuring sharp waves and oscillatory population activity with the genetically encoded calcium indicator GCaMP6f. Front Cell Neurosci 13: 274.
  15. Maier, N., Morris, G., Johenning, F. W. and Schmitz, D. (2009). An approach for reliably investigating hippocampal sharp wave-ripples in vitro. PLoS One 4(9): e6925.
  16. Maier, N., Nimmrich, V. and Draguhn, A. (2003). Cellular and network mechanisms underlying spontaneous sharp wave-ripple complexes in mouse hippocampal slices. J Physiol 550(Pt 3): 873-887.
  17. Miyawaki, T., Norimoto, H., Ishikawa, T., Watanabe, Y., Matsuki, N. and Ikegaya, Y. (2014). Dopamine receptor activation reorganizes neuronal ensembles during hippocampal sharp waves in vitro. PLoS One 9(8): e104438.
  18. Oakley, H., Cole, S. L., Logan, S., Maus, E., Shao, P., Craft, J., Guillozet-Bongaarts, A., Ohno, M., Disterhoft, J., Van Eldik, L., Berry, R. and Vassar, R. (2006). Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer's disease mutations: potential factors in amyloid plaque formation. J Neurosci 26(40): 10129-10140.
  19. Schlingloff, D., Káli, S., Freund, T. F., Hájos, N., Gulyás, A.I. (2014). Mechanisms of sharp wave initiation and ripple generation. J Neurosci 34(34):11385-11398.
  20. Sun, Z. Y., Bozzelli, P. L., Caccavano, A., Allen, M., Balmuth, J., Vicini, S., Wu, J. Y. and Conant, K. (2018). Disruption of perineuronal nets increases the frequency of sharp wave ripple events. Hippocampus 28(1): 42-52.
  21. Tanaka, Y., Furuta, T., Yanagawa, Y. and Kaneko, T. (2008). The effects of cutting solutions on the viability of GABAergic interneurons in cerebral cortical slices of adult mice. J Neurosci Methods 171:118-125.
  22. Ting, J. T., Daigle, T. L., Chen, Q. and Feng, G. (2014). Acute brain slice methods for adult and aging mice: application of targeted patch clamp analysis and optogenetics. Methods Mol Biol 1183:221-242.
  23. Ting, J. T., Lee, B. R., Chong, P., Soler-Llavina, G., Cobbs, C., Koch, C., Zeng, H. and Lein, E. (2018). Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method. J Vis Exp (132):53825.
  24. Wilson, M. A. and McNaughton, B. L. (1994). Reactivation of hippocampal ensemble memories during sleep. Science 265(5172): 676-679.
  25. Wu, C., Luk, W. P., Gillis, J., Skinner, F. and Zhang, L. (2005). Size does matter: generation of intrinsic network rhythms in thick mouse hippocampal slices. J Neurophysiol 93(4): 2302-2317.
  26. Ye, J. H., Zhang, J., Xiao, C. and Kong, J. Q. (2006). Patch-clamp studies in the CNS illustrate a simple new method for obtaining viable neurons in rat brain slices: glycerol replacement of NaCl protects CNS neurons. J Neurosci Methods 158(2):251-259.

简介

[摘要]我们描述了一种制备急性脑切片的方案,该方案可在体外产生强大的海马锐波波纹(SWR)。该协议经过优化,其简单性和可靠性可用于制备溶液,切片和恢复孵育。通过该协议,几乎每只小鼠中的大多数切片都表现出强烈的自发性SWR,持续约24小时,而“标准”低钠切片协议的生存力为20-30%。SWR是海马的自发性神经元活动,对于巩固发作性记忆至关重要。可靠表达SWR的脑片可用于研究离体的记忆障碍和脑退化疾病实验。SWR的自发表达对记录期间的切片和灌注/充氧条件敏感。SWR的振幅和丰度通常用作可行切片的生物标记。关键改进包括快速循环,切片后较长的恢复时间(3-6小时)以及使组织在32岁时恢复°C在充分灌注的培养箱中。我们定制设备中的切片可以表达自发SWR数小时,这表明在本地网络中具有平衡的激发和抑制作用的时间很长。年龄较大的小鼠(约产后180天)的切片显示出与年龄较小的小鼠(产后21-30)相似的生存力。


[背景]急性脑切片已成为电生理学和其他神经科学研究的重要准备。尽管有大量文献描述了脑切片的用法,但通常不那么详细地讨论可靠地生产可行切片的方案。来自年长动物的切片通常不太可行。通常使用健康的细胞形态和神经元的电生理特性来描述脑切片的活力。但是,组织中看起来健康的神经元并不总是转化为正常的网络行为。例如,用标准蔗糖替代方案制备的薄片(例如,Aghajanian和Rasmussen ,1989 ;Ye等人,2006)为形态研究提供了形态学上健康的细胞,但不表达自发的网络活动,例如海马尖波波纹(SWR)。

SWRS的睡眠和安静restfulness期间发生在海马自发的神经元群的事件(综述见Buzs áき,2015年)。诸如探索环境之类的经验在海马中被记录为神经元组件(也称为“位置细胞组件”)的顺序激活。然后,这些序列在睡眠或安静状态(离线状态)期间在SWR中重新激活,并有助于记忆的巩固(Wilson和McNaughton,1994; Lee和Wilson,2002; Ji和Wilson,2007)。SWR的抑制削弱了海马依赖性记忆(Ego-Stengel和Wilson ,2010),这表明SWR对于在缺乏相关感觉输入的情况下对于与神经元集合相关的体验的重新激活很重要。

自发性SWRs也在体外在海马切片中发生(Kubota等人,2003 ; Maier等人,2003和2009; Colgin等人,2004; Behrens等人,2005; Miyawaki等人,2014; Keller等人。,2015)。然而,在标准的浸没式记录室中,由于灌注不足,很少见到SWR。对切片的两侧剧烈灌注似乎是用于提供足够的氧气,其允许要发生的SWRS的必要条件(吴等人,2005; ħ á 。书等人,2009;迈尔。等人,2009)。

根据我们的经验,用标准的高蔗糖低钠方案制备的切片(Aghajanian和Rasmussen ,1989)仅具有较低的表达自发SWR的可能性(20-50%)。出生后(P)超过30天的小鼠的切片表达SWR的可能性进一步降低,通常无法诱导LTP。切片后延长恢复时间通常会有所帮助。具有一致(迈尔等人,2009),我们还观察到,SWRS按常规切片培养室,其具有流体无源对流或氧气气泡引起的对流(很少表达例如,脑片门将-4装置,自动化Scientific)中。用小泵强制循环流体显着增加了SWR的表达。我们已经测试了35只P 35至P180天龄小鼠的198片,每只测试动物均显示自发性SWR,包括6个月大的带有明显淀粉样蛋白斑块的5xFAD小鼠和来自遭受严重创伤性小脑损伤的小鼠的切片。

快速循环可能很重要,原因有两个:1.切成薄片(350-500 μ米)是用于保存SWR生成电路是必不可少的。切片两侧的快速循环可将氧气输送速度提高到组织的厚度中。2. SWR是每秒发生1-2次的自发活动。大量神经元的持续激活需要快速循环提供更多的氧气。

我们的协议还旨在简化操作,即使用储备溶液以最大程度地缩短溶液制备时间(<30分钟),使用简单的低钠高蔗糖切割溶液,而不使用诸如NMDG的不稳定化合物(Tanaka等,2008)和抗氧化剂( Brahma等,2000),并省略了心脏内灌注和保护性恢复方案的复杂程序(Ting等,2014和2018)。我们已经使用该协议发表了一些研究论文(Jiang等人,2018; Sun等人,2018; Li等人,2019)。

关键字:脑片, 海马体, 锐波波纹, 电生理学, 振动, 脑组织活力

材料和试剂 

纸巾

用于局部场电势电极的玻璃吸管,外径1.5 mm x内径1.1 mm,长7.5 cm的带有内部细丝的硼硅酸盐玻璃吸管(Sutter I仪器,目录号:BF150-110-7.5)
尼龙网(组件供应UMN-600),粘在切片支架的底部
硅胶管(1 / 4-3 / 8,VWR,货号:89403-862),用于连接泵和顶部腔室
一次性移液管(Thermo Scientific,Samco 3.2 ml),用于在培养室和记录室之间进行切片转移
动物
使用野生型小鼠(C57BL / 6J,RRID:IMSR_JAX:000664,Jackson Labs)和转基因5xFAD小鼠(RRID:MMRRC_034840-JAX,Jackson Labs)(Oakley等,2006)。实验小鼠来自我们的乔治敦(Georgetown)殖民地,按标准的12小时光照/黑暗周期饲养,随意提供食物和水,所有实验程序均按照乔治敦大学动物保健和使用委员会的指导进行。 超级胶(液态,乐泰,目录号:1775049),用于在切片过程中粘合大脑
蔗糖(ACS R试剂,Fisher Scientific ,目录号:S5-500)
氯化钾(ACS R eagents,Fisher Scientific ,CAS :7447-40-7)
MgSO 4 (ACS R试剂,Fisher Scientific ,CAS :7487-88-9)
CaCl 2 (ACS R试剂,Fisher Scientific ,目录号:C79-500)
葡萄糖(ACS R试剂,Sigma-Aldrich ,目录号:G8270-5KG)
NaH 2 PO 4 (ACS R试剂,Sigma-Aldrich ,目录号:S0751-1KG)
HEPES(ACS R代理,Fisher Scientific ,目录号:BP310-1)
NaCl(ACS R试剂,Sigma-Aldrich ,目录号:S5886-10KG)
卡波金(95%氧气2 ,5%CO 2 ,罐和可用的调节器从本地天然气供应商)
NaHCO 3粉末(ACS R试剂,Fisher Scientific ,目录号:S233-500)
切片切割ACSF解决方案(请参阅食谱)
库存1:蔗糖 库存2:切割ACSF 库存3:NaHCO 3粉末 孵育/记录ACSF(见ř ecipes)


设备   

切片准备 振动切片机(Leica ,型号VT1000S)

水浴(例如,西南科学,型号:SH W10LD 10升浴)
数字温度计(VWR可追溯双温度计,目录号:23226-656,或任何带0.1的类型° C精度)
图2中所示的定制孵育室由1/4有机玻璃(丙烯酸)板制成,购自McMaster-Carr
从具有1/2 x 1/2 x 1/2格的蛋箱光扩散板切割切片架(e-Plastics SKU:W / EGG.500X2X4)
600毫升玻璃烧杯(VWR ,目录号:89001-078)
需要三台鼓风机(例如,来自亚马逊的气石气瓶气泡扩散器)(两台用于培养室的孵化用气体鼓风机和记录ASCF烧瓶,一台用作记录室中ACSF循环的过滤器)
低压,低流量泵,可通过太阳能微型喷泉/鸟巢套件(Amazon)获得。该泵提供了〜1的流程,000个毫升/分钟,并且使用一个安全的低电压电源(下)
泵的可变直流电源(电路专家CSI5003XE或B&K Precision 1550直流电源)
小鼠断头手术剪刀(WPI ,目录号:501743G)
弹簧剪刀(WPI ,目录号:15905G),用于穿过颅骨并拔出大脑
尺寸4水彩笔尖刷(办公室仓库596713),用于转移脑片
杜蒙#7学生钳(精细的小号cience牛逼ools ,目录号:97797-00),用于提取大脑
锥形刮铲(PTFE涂层,VWR ,目录号:10806-412),用于拔除大脑
杜蒙#5学生镊子(精细小号cience Ť ools ,目录号:91150-20),用于调节在记录室切片位置
  用于SWR录制 尽管大多数电生理设备都可用于记录SWR,但必须以高循环速率灌注切片的两侧。见Maier等。(2009)和ħ á 。书等。(2009年)修改了补丁记录台上的传统暗室。以下列出的是低成本本地现场电势记录系统的部件。该系统易于使用且可靠,可一次记录多个小时的SWR。 局域电位记录室
可灌注切片两侧的腔室很重要。请参阅过程和数据记录部分。图3说明了定制记录室中的流向。 LFP放大器,带有探测头级的1000倍放大(例如,Warner Instruments ,型号:DP 311A)
数据采集系统以数字化的数据(例如,NeuronNexue智能箱,允许多个信道与一个电极阵列)
示波器(例如,Digikey ,用于定位电极2250-SDS1022-ND) 
用于放置电极的音频监视器(AM S ystems 3300)
手动微机械手(SD I仪器SKU MX160)和用于固定机械手的磁力架(SD I仪器)
625 nm LED(Thorlabs ,型号:M625D3),用于在腔室下方进行切片照明
移液器拉拔器(Sutter ,型号:P97),用于拉动超低阻抗移液器以进行局部场电势(LFP)记录
LFP记录和刺激电极(由移液器拔出器拉出的低阻抗(〜500K)玻璃移液器)。低电极阻抗(50-500 K )对于低噪声记录很重要。
用于ACSF循环的蠕动泵(例如,Pulsatron Dolphin系列蠕动计量泵,60.0 gpd / 25 PSI,Norprene管,0.25“ ID x .44” OD)。
用于连接泵和记录室灌注的塑料管(VWR ,目录号:89403-850,3 / 32-5 / 32 Tygon管)
管夹流量调节器(VWR达隆®普通Hosecock夹),对于记录室的调节灌注率
1000 ml玻璃烧瓶,用于循环记录室的ACSF灌注(VWR ,目录号:75804-652)
奥林巴斯,尼康或美国显微镜提供的立体变焦解剖显微镜,10-40倍变焦(SKU:V-SM-3 SD I仪器)
记录台(纽波特,目录号:IG-22-2)

这个2 x 2英尺的光学面包板可以安装在标准实验室工作台上。从SWR记录LFP不需要隔振表。如果使用低阻抗玻璃电极并且在设置的30英寸范围内没有仪器电源线,也可以避免使用法拉第笼。   


程序 

制备

切片之前,准备两个人工脑脊髓液(ACSF)从储备溶液(在下面的配方部分程序)的解决方案:所述的切削ACSF(200毫升)和温育ACSF(2 ,000毫升)。将两者都用碳气剧烈鼓泡约3分钟。然后将切割ACSF冷却至4 ℃,在冰/水浴中,并温育ACSF温热至32 ℃,在培养装置(见配方细节小号部分)。   切片
严格按照NIH指南,严格按照乔治敦大学医学中心机构动物护理和使用委员会(IACUC)批准的协议使用动物。
通过自由呼吸异氟烷蒸气(> 5%)约20 s,在麻醉罐(由IACUC批准)中对动物进行深度麻醉。完全麻醉后(肌肉张力消失,对尾巴捏紧无反应),用一把外科剪刀将动物迅速断头。
用一把小手术剪刀沿着头皮的中线切开皮肤,露出头骨,然后将头浸入冷的(4°C)ACSF切割溶液中约1分钟。浸入水中时,取出覆盖颅骨的组织以减少隔热。此步骤后,应将大脑冷却至5-10 °C 。
如图1所示,用一把小外科剪刀剪开头骨。使用Dumont#7镊子去除大脑上方的头骨碎片。从头端开始用刮铲轻轻地抽出大脑,并在约4°C下让大脑滑入切割的ACSF溶液中。
重要提示:确保刮铲的尖端位于大脑腹侧的中线,以避免接触腹颞叶,否则可能导致腹侧海马受压。切削ACSF的温度必须高于0 °C,以避免低温引起的损坏。  
图1.用于脑部提取的头骨切割线。红线标记穿过头骨的切口。数字是建议的顺序。切口5不能太深,以确保不会干扰切口下方的皮质组织。在拔脑之前,需要将头部冷却至10 °C以下。只要大脑是冷的,它对于短时间的提取并不是至关重要的。在提取过程中,应注意不要拉伸或压缩海马体。   在震动刀切割平台的表面上涂上一层薄薄的超级胶水,以便在平台上粘合大脑。
使用抹刀拿起从切削液中的脑(脑涨的背侧),并使用小片纸巾的从腹侧吸收过量的溶液,然后将脑的腹侧放到吨他切割板的表面涂有超级胶液。
在大脑的背面施加少量压力,以确保大脑和切割台之间牢固的胶粘接触。
现在,将切割台安装在振动切片机上,并用4 °C的切割ACSF填充切割台的腔室。从头端到尾端以水平方向切开大脑。与水平平原之间的倾斜角度为12.7度(在鼻尖处较高),可以更好地保留CA3和CA1区域之间的连接,从而更好地表达SWR(Miyawaki等人,2014)。然而,当在切480-500 μ米,12.7度的倾斜角的并不重要。较厚的切片(> 350 μ米,迈尔等人,2009 )是用于SWRS的表达很重要。
海马切片(即我们切断488 μ米)在高振动速度(〜70赫兹)和低前述速度(1-2在振动切片机设置)被切割。切割时允许ACSF切割溶液的温度从〜4°C升高到10°C是可以的。如果总切割时间超过30分钟,则需要用碳素起泡,以防止切割溶液的pH值发生变化。
每次切割后,将切片用细刷轻轻转移到培养室中的培养ACSF中,同时注意不要弯曲或压缩组织。在传输之前,可以将整个水平切片(两个半球)修剪成两半。通常,我们从每只动物获得4-5个完整的水平切片(从两侧各取10个海马切片)。
  孵化
在恒温装置(图2)中以恒定的流量循环将孵育温度保持在32°C。调整可变电源并保持〜1的比率,000毫升/分钟(3.5-4 V至泵)。流速应不高于1 ,000毫升/分钟,以减少在这可能会导致对组织的机械损伤的溶液运动或浮动的切片。
应在烧杯中以200-400 ml / min的速度不断向鼓泡中通入二氧化碳,以保持ACSF溶液中O 2 / CO 2饱和。
在32 °C下2 h后,将水浴温度降低到27°C,并让ACSF从32°C缓慢冷却到27°C(大约需要1.5 h)。继续孵育直至使用切片。切片在孵育设备中可存活约24小时。
 
图2 。培养箱的构造。该设备的上部容器是由丙烯酸塑料玻璃制成的流动ACSF容器。容器内部有一个切片保持室(从蛋箱式灯扩散器切下[请参见设备] )。将切片保持在分开的隔室中(顶视图),然后浸入循环的ACSF溶液中。尼龙网(请参见材料)粘在切片支架上,以实现良好的流通。切片保持器的底侧具有比顶侧更高的流速,其在每个隔室中提供小的向下电流以使切片抵靠网状物稳定。顶部容器中的屏障可保持液位。仪器下方是一个600毫升的玻璃烧杯,它坐在水浴中,水深约6厘米,以保持32 °C的温度。将烧杯中的ACSF以200-400 ml / min的速度连续通入碳素。低压泵(请参阅设备)用于循环ACSF。泵由可变直流电源供电(请参阅设备)。调整泵的电压(3-4 V),使流量达到〜1,000 ml / min(约为泵容量的1/2)。总流体体积是800-1 ,000毫升。盖好上部容器,以减少许多小时后的蒸发和摩尔渗透压浓度变化。   SWR记录
建立孵育/记录ACSF溶液的循环在记录室:将800-1 ,000毫升孵育ACSF入1 ,000个烧瓶中,把烧瓶内的货架上的记录室的水平1-1.5米以上允许重力流通过3 / 32x 5/32 Tygon管将其从烧瓶虹吸到反应室中。气石连接到烧瓶中的油管末端,并用作用于循环的流体过滤器,并用作油管的锚固件,以留在烧瓶的底部。用实验室真空吸尘器或注射器抽吸虹吸管。流量由油管夹调节。正常循环速度为40-100毫升/分钟。从记录室中滴落的液体收集在位于该室下方的100 ml烧杯中,并通过蠕动泵送回烧瓶。我们对ACSF的进,出口流都使用了重力,因为这可以完全消除在运行蠕动泵时挤压管路所产生的静电噪声。烧瓶中的ACSF以50-100 ml / min的速度连续不断地通入碳素,以保持O 2 / CO 2饱和
孵育> 3小时后,SWR逐渐出现。孵育7小时后,大多数切片应从海马的最背侧到腹侧大部分表达SWR。
要将切片放置在记录室中:用流体移液管(将移液管的小端切掉)小心地将切片从培养室移入记录室的中心。切片应通过流体的垂直流动在腔室的中央保持稳定。切片的位置和方向可以通过一对#5细镊子轻轻调节。要特别注意不要弯曲或压缩组织。对组织的机械干扰可以使SWR停止或衰减几分钟。海马应以其全变焦放大倍数(40 x )放置在体视显微镜视野的中心。
准备记录电极:使用P-97拉拔器通过5-7次拉动拉低电阻玻璃电极。多个拉导致50-100的电阻ķ欧姆当充满ACSF,和末端开口为约10 μ米。低电阻导致LFP记录的低噪声。
将电极安装到放大器的头级。将电极的尖端插入海马切片顶部上方记录室的液体表面。此步骤无需显微镜即可完成。打开示波器和音频监视器。示波器的基线应该平坦,音频监视器的静电噪声应该很小。基线跳动和较大的音频噪声表明电极填充不良(气泡)。测量电极的电阻,应小于100 K欧姆。
放置电极头的方法:将显微镜的变焦放大倍数调到最小(0.75 x ),并调节焦点以找到电极头。用操纵器调整吸头的位置,以降低吸头的位置,使其接近记录位置(CA1的层半径,图4A)。调整电极时,请始终调整显微镜焦点,以使电极尖端清晰可见。如果电极的尖端不在显微镜的焦点内,切勿移动电极。此时,请勿将尖端插入组织。现在增加显微镜的变焦倍率到2-3 X ,调整焦距找到电极尖端。在高放大倍率下,继续降低电极,直到其位于组织表面上(切片表面和电极头均处于聚焦状态)。现在,在音频监视器中应该可以听到波纹振荡的声音(〜100 Hz )。
要将电极头插入组织中:缓慢将电极头推进到组织表面。接近组织时,涟漪声变大。当尖端接触组织时,音频监视器中会听到很大的声音。在接触位置,慢慢转动机械手的插入旋钮轴1 / 4-1 / 2圈,其前进尖端90-180 μ米到组织中。请注意,电极的机械干扰会在几分钟内衰减SWR的幅度,因此缓慢的插入非常重要(90秒钟约20秒)μ米提前)。显微镜无法观察到电极头的这种小而缓慢的移动,因此需要通过音频监视器进行引导。后几分钟电极插入UTES,剧烈具有高振幅的SWRS应出现并持续几个小时(图4)。我们通常记录100-200 μ米表面的下方,其中的信号是最大的。
背侧海马的切片的SWR速率比腹侧海马的切片慢得多。仅当切片的两面在浸没式记录室中充分灌注时,SWR才能维持。SWR在常规的单侧灌注室(例如Warner RC-22)中不能很好地表达。
记录期间SWR的维持:在图3所示的两侧灌注室中,SWR可以维持约24小时(图4C,图5)。
 
图3.录音室。示意图显示了一个定制的记录室,该记录室在切片周围产生了向下的垂直流体流。流动向切片的两侧提供有力的灌注。同样,向下流动可保持切片稳定(无需在切片上施加重量)。我们使用屏障来维持组织顶部的液位。ACSF的出口是重力滴,可显着降低由真空抽吸引入的电噪声。同样,ACSF可以通过蠕动泵收集并再循环。腔室底部的发光二极管(LED)芯片用于提供漫射照明。使用红色LED(630 nm)可以避免表达通道视紫红质2的神经元的光激活。红光还可以增加厚组织的透明度。该腔室很厚,无法安装在复合显微镜上以修补细胞。但是,SWR在常规的单侧灌注室(例如Warner RC-22)中不能很好地表达。确保组织的两侧被灌注孔是非常重要的(ħ á 。书等人,2009 ;迈尔。等人; 2009年Caccavano等人的SWRS到在单元贴片的录音室表达很好,2020)。  
图4.年轻和年老小鼠的SWR。A.左:通过超低电阻的玻璃移液管(<100K欧姆,充满生理盐水)从纹状体半径记录SWR ,可提供出色的信噪比。右迹线:在幼小的小鼠中观察到大量的SWR,具有高的尖波振幅(上迹线)和波纹振荡(底部插入物,为清楚起见在60-180 Hz之间过滤)。B.所有测试的老年小鼠(P6月,四分之四)均表达SWR。与幼鼠相比,SWR的振幅和比率更低。C.来自B中同一动物切片的SWR,在储藏室中过夜灌注后记录。虽然幅度和重复率进一步降低,但驻波比的出现率并未出现大的下降。   数据分析   SWRs的发生率,LFP振幅和钙信号可以将其与癫痫发作/发作间期峰值区分开来(Li等,2019)。SWR的发生率很高(1-3个事件/秒),而尖峰间的峰值要慢得多(0.3-0.01个事件/秒)。SWR还伴有〜100 Hz的纹波振荡(图4 A插图),但壁尖峰大多伴有低伽马(20-50 Hz)。   培养箱中的回收对于SWR表达很重要。根据我们对198个切片的经验,在恢复时间少于2小时的切片中从未见过SWR,但是在恢复时间7小时后才看到。通常,较年轻的小鼠(P28-P35)会更快恢复,并且孵育3-4小时就足够了。年龄较大的小鼠(P60-P180)需要更长的时间才能从切片中恢复,但是5-7小时后,大多数切片显示出SWR。在用GCaMP-6f基因编码的动物中,切割后海马区显示出强烈的绿色荧光,这表明细胞内钙的浓度升高。在培养箱中恢复数小时后,荧光逐渐降低。在常规的烧杯和氧气鼓风机式培养箱中,通常只有一小部分切片可以很好地恢复并表达SWR。传统腔室中的流体循环没有得到很好的控制。当氧气输送的优化程度较低时,组织的活力会随着切片创伤的恢复而下降。如果ACSF循环受到影响,更长的恢复时间不会提高生存能力。   为了改善恢复期的流体循环,我们制造了许多定制的培养槽以延长恢复时间。这些腔室的共同特点是通过搅拌棒或泵进行强制循环。我们发现普通蠕动泵不能为容纳2-3只动物的〜26片的大型孵化室提供足够的流量。因此,我们使用了低压直流泵(从太阳能喷泉获得),流量约为1000毫升/分钟。如此高的流量大大改善了ACSF在所有切片周围的循环。更好的氧合还可以使切片在更高的温度(〜32°C)下恢复。该设备为从切割相关的细胞损伤和细胞内钙升高中恢复提供了稳定的条件。我们已经发表了使用厚切片和强制循环的部分工作(Sun等人,2018 ; Jiang等人,2018 ; Li等人,2019)。从旧的(P3-6月)切片的动物也有尽管创伤性脑损伤(未发表的观察),或在动物5XFAD发展淀粉样蛋白斑(表观正常SWR表达制备由这个协议(图4B / C)Caccavano等人。,2020 )。   在大多数切片中,SWR的幅度在记录室中随时间逐渐减小。在记录室中,SWR振幅花了大约5小时才能减小一半(图5)。看来6个月大的老鼠比一百万岁大的老鼠的振幅降低更多。长时间记录后,SWR幅值似乎变化不大。可以看到更多具有相似振幅的S WR(图5)。 似乎没有自发SWR的切片在诱发的活动中也表现出较差的表现,例如,当沐浴在含有20mg / mM的卡巴胆碱(20 mM)中时,破伤风刺激(100 Hz x 100脉冲)不能增强和theta-γ振荡的低振幅。ACSF。这些不太可行的切片通常在切割后需要较短的恢复时间(未发表的观察结果)。   切片“生存力”的生物标记是一个重要的问题。在此手稿中,如果SWR长时间表达强烈,我们认为切片是可行的。表达SWR需要网络完整性以及突触激发/抑制的平衡(Schlingloff等,2014)。  
图5.记录时间和幅度下降。A.连续记录(> 10 h)一片P30动物的SWR振幅。切片后10小时开始记录。SWR振幅在整个周期内逐渐减小。还原速度在5小时内降至一半。B.切割后10和23 h的痕迹。请注意,振幅较小的SWR在数小时后显示出不成比例的减少。在该图中,我们显示从SWR信号纹状体东方明珠Oriens (信号极性从该逆转图4)。   菜谱   切片ACSF溶液(每个实验200毫升,使用前由两种储备溶液和0.43克NaHCO 3粉末制成)
最终组成:蔗糖(252.0 mM);氯化钾(3.0毫米); MgSO 4 (4.0 mM);CaCl 2 (1.0 mM);葡萄糖(10.0 mM); NaHCO 3 (10.0 mM);NaH 2 PO 4 (1.25 mM);HEPES(5.0 mM);充满了生碳素;磷= 7.4。   库存1:蔗糖 177 g蔗糖,溶于1,000 ml热(65 °C )蒸馏水中
然后添加蒸馏水至最终体积为2,000 ml
使用前将原料保持在4 °C
2,000 ml储备液可用于10个实验(每个实验180 ml),保持在4 °C且可在一个月内使用。   库存2:切割ACSF 按以下顺序将化学品溶解在180毫升蒸馏水中
200毫升试剂(分子量)克 氯化钾(74.55)0.45克 MgSO 4 (120.37)0.96克 葡萄糖(180.8)3.60克 NaH 2 PO 4 (119.98)0.30克 HEPES(238.3)2.38克 CaCl 2 ·2H 2 O(147.02)0.29克 所有化学品完全溶解后,加蒸馏水至终值200毫升
使用前,请将库存保存在4 °C的冰箱中
200 ml储备液可用于10个实验(每个实验20 ml,保持在4 °C并在一个月内使用)。   库存3:NaHCO 3粉末 准备10份0.43克NaHCO 3粉末(每个储存在1.5毫升Eppendorf管中)以进行10次实验 这样可以大大减少每次实验之前的准备时间   每次实验前制作200毫升切片ACSF溶液: 取180 ml的蔗糖原液,然后加入20 ml的切削原液,使最终体积达到200 ml。用磁力搅拌棒将两种溶液剧烈搅拌混合。
将NaHCO 3粉末放入一个Eppendorf管(0.43 g)中,放入200 ml切片ACSF中。缓慢搅拌下缓慢加入粉末,直至所有NaHCO 3细晶体溶解。用鱼缸中的空气石吹入碳素3分钟,以确保O 2 / CO 2完全饱和。使用前将溶液冷却至4 °C 。
重要提示:使用前测量切削液的温度。切片的最佳温度为3-6 °C 。由于两种储备溶液在混合前均处于4 °C的温度,因此只需约10分钟即可冷却ACSF切割溶液。 孵育ACSF(2 ,000毫升每每个实验中,从原液下面和4.37克碳酸氢钠制成3粉末)。
孵育ACSF的最终组成:NaCl(132.0 mM); 氯化钾(3.0毫米); MgSO 4 (2.0 mM);CaCl 2 (2.0 mM);葡萄糖(10.0 mM); NaCO 3 (10.0 mM);NaH 2 PO 4 (1.25 mM);HEPES(5.0 mM)。与卡波金饱和的,p H = 7.4,渗透压305-310   孵化/记录ACSF
孵化/记录ACSF的储备液 按以下顺序将化学品溶解在1,600毫升蒸馏水中
2,000毫升试剂(配方重量)克 氯化钠(58.44)    144.0克 氯化钾(74.55)    4.5克 MgSO 4 (120.37)    4.8克 葡萄糖(180.8)    36.0克 NaH 2 PO 4 (119.98)    3.0克 HEPES(238.3)    23.8克 CaCl 2 ·H 2 O(147.02)    5.9克 所有化学品完全溶解后,添加蒸馏水至最终体积为2,000 ml
将储备溶液保持在4 °C,并在一个月内使用。2 ,000毫升ACSF原液是良好的10个实验
准备20份4.37克NaHCO 3粉末(每个储存在17毫升带帽培养管中)以进行20个实验。这样可以大大减少每次实验之前的准备时间
  每次实验前将2,000 ml培养液放入ACSF中: 取200 ml培养的ACSF储备液并溶于1600 ml温暖的(35 °C )蒸馏水中
用磁力搅拌棒搅拌溶液
将一管NaHCO 3粉末(4.37 g)加入溶液中,同时剧烈搅拌直至NaHCO 3细晶体完全溶解
加入温热的(35 °C )蒸馏水,使最终体积达到2,000 ml
用碳纤维起泡3分钟,以确保O 2 / CO 2完全饱和

注意:测量ACSF的温度,在将其装入培养箱之前,应为〜32 °C 。   致谢   由NIH R03AG061645,乔治城大学医学中心院长的Toulmin赠款支持,2017财年,2019财年。  

  利益争夺   

作者宣称没有相互竞争的经济利益。  

 

伦理   

严格按照NIH指南,严格按照乔治敦大学医学中心机构动物护理和使用委员会(IACUC)批准的协议使用动物。


参考文献  

Aghajanian ,GK和Rasmussen,K。(1989)。面部细胞核的细胞内研究说明了在成年大鼠脑切片中获得活的运动神经元的简单新方法。 突触3(4):331-338。

Behrens,CJ,van den Boom,LP,de Hoz ,L.,Friedman,A. and Heinemann,U.(2005)。体外诱导尖波波纹复合体和海马网络的重组。Nat Neurosci 8(11):1560-1567。
布拉马(Brahma,B.),佛曼(Forman),稀土(RE),斯图尔特(EE),EE,尼科尔森(C. 抗坏血酸抑制脑片水肿。Ĵ神经化学杂志74:1263至1270年。
Buzs áき,G.(2015)。海马尖波状波纹:情景记忆和计划的认知生物标志物。海马25(10):1073-1188。 
Caccavano ,A.,Bozzelli ,PL,Forcelli ,PA,Pak,DTS,Wu,JY,Conant,K.和Vicini ,S.(2020)。在家族性阿尔茨海默氏病小鼠模型中,在海马锐波波纹期间,抑制性小白蛋白篮子细胞的活性选择性降低。Ĵ神经科学DOI :10.1523 / JNEUROSCI.0425-20.2020。
Colgin ,LL,Kubota,D.,Jia,Y.,Rex,CS and Lynch,G.(2004年)。大鼠海马切片中产生自发性尖锐波的长期增强能力受损。Ĵ生理学558(PT 3):953-961。
Ego-Stengel,V.和Wilson,MA(2010)。休息期间中断与涟漪相关的海马活动会损害大鼠的空间学习。海马20(1):1-10。
ħ á乔斯,N.,Ellender ,TJ,Zemankovics ,R.,曼,EO,埃斯里,R.,克拉格,SJ,弗氏,TF和保尔森,O.(2009)。维持淹没海马切片中的网络活动:供氧的重要性。Eur J Neurosci 29(2):319-327。
Jiang H.,Liu,S.,Geng ,X.,Caccavano ,A.,Conant,K.,Vicini ,S.和Wu,J.(2018)。用弱电刺激使海马尖波起搏。前沿神经科学12:164。
Ji,D. and Wilson,MA(2007)。睡眠期间视觉皮层和海马的协调记忆重播。Nat Neurosci 10(1):100-107。
Keller,MK,Draguhn ,A.,M.和Reichinnek ,S.(2015)。小鼠海马装配体的活性依赖可塑性在体外。前神经回路9:21 。
Kubota,D.,Colgin ,LL,Casale ,M.,Brucher ,FA和Lynch,G。(2003)。海马切片中的内生波。Ĵ神经生理学89(1):81-89。
Lee,AK和Wilson,MA(2002)。慢波睡眠期间海马体中顺序性经历的记忆。神经元36(6):1183-1194。
李平,耿晓星,江华,卡卡瓦诺,A.维奇尼,S.和吴建元(2019)。用遗传编码的钙指示剂GCaMP6f测量尖波和振荡种群活动。前沿细胞神经科学13:274。
Maier,N.,Morris,G.,Johenning ,FW和Schmitz,D.(2009)。一种可靠地体外研究海马尖波波纹的方法。PLoS One 4(9):e6925。
Maier,N.,Nimmrich ,V。和Draghn ,A。(2003)。小鼠海马切片中自发的尖峰波状复合体的细胞和网络机制。Ĵ生理学550(PT 3):873-887。
Miyawaki,T.,Norimoto ,H.,Ishikawa,T.,Watanabe,Y.,Matsuki ,N.和Ikegaya ,Y.(2014)。多巴胺受体的激活在体外海马尖波期间重组神经元集合。PLoS One 9(8):e104438。
Oakley,H.,Cole,SL,Logan,S.,Maus,E.,Shao,P.,Craft,J.,Guillozet-Bongaarts ,A.,Ohno,M.,Disterhoft,J.,Van Eldik ,L ,Berry,R。和Vassar,R。(2006)。在具有五个家族性阿尔茨海默氏病突变的转基因小鼠中,神经内β-淀粉样蛋白聚集,神经变性和神经元丢失:淀粉样蛋白斑形成的潜在因素。Ĵ神经科学26(40):10129-10140。
Schlingloff ,D.,Káli ,S.,Freund,TF,Hájos ,N.,Gulyás ,AI(2014)。尖波引发和波纹产生的机制。Ĵ神经科学34(34):11385- 113 98。
Sun,ZY,Bozzelli ,PL,Caccavano ,A.,Allen,M.,Balmuth ,J.,Vicini ,S.,Wu,JY和Conant,K.(2018)。神经周神经网的破坏增加了尖波波纹事件的发生频率。海马28(1):42-52。
田中(Y.)田中(Tanaka),古田(T.Furuta ),柳川(Yanagawa )和T. 切割液对成年小鼠大脑皮质切片中GABA能神经元活力的影响。Ĵ神经科学方法171:118-125。
Ting,JT,Daigle,TL,Chen Q.和Feng,G.(2014)。成年和衰老小鼠的急性脑切片方法:靶向膜片钳分析和光遗传学的应用。方法分子生物学1183:221 - 242。
Ting,JT,Lee,BR,Chong,P.,Soler- Llavina ,G.,Cobbs,C.,Koch,C.,Zeng,H. and Lein ,E.(2018年)。使用优化的N-甲基-D-葡萄糖胺保护性恢复方法制备急性脑切片。J Vis Exp (132):53825。
马萨诸塞州威尔逊(Wilson,MA)和麦克诺顿(McNaughton,BL)(1994)。睡眠期间恢复海马合奏记忆。科学265(5172):676-679。
Wu,C.,Luk ,WP,Gillis,J.,Skinner,F. and Zhang,L.(2005年)。大小很重要:在厚厚的小鼠海马切片中生成内在的网络节律。Ĵ神经生理学93(4):2302年至2317年。
Ye,JH,Zhang,J.,Xiao C. and Kong,JQ(2006)。中枢神经系统的膜片钳研究说明了一种在大鼠脑切片中获得存活神经元的简单新方法:甘油替代氯化钠可保护中枢神经系统神经元。J Neurosci方法158(2):251-259。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
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: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用:Liu, L., Zhou, X. and Wu, J. (2020). Preparing Viable Hippocampal Slices from Adult Mice for the Study of Sharp Wave-ripples. Bio-protocol 10(19): e3771. DOI: 10.21769/BioProtoc.3771.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。