参见作者原研究论文

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May 2016
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Antisense Oligodeoxynucleotide Perfusion Blocks Gene Expression of Synaptic Plasticity-related Proteins without Inducing Compensation in Hippocampal Slices
反义寡脱氧核苷酸灌注可阻突触可塑性相关蛋白在海马切片上的基因表达且无诱导补偿作用   

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Abstract

The elucidation of the molecular mechanisms of long-term synaptic plasticity has been hindered by both the compensation that can occur after chronic loss of the core plasticity molecules and by ex vivo conditions that may not reproduce in vivo plasticity. Here we describe a novel method to rapidly suppress gene expression by antisense oligodeoxynucleotides (ODNs) applied to rodent brain slices in an “Oslo-type” interface chamber. The method has three advantageous features: 1) rapid blockade of new synthesis of the targeted proteins that avoids genetic compensation, 2) efficient oxygenation of the brain slice, which is critical for reproducing in vivo conditions of long-term synaptic plasticity, and 3) a recirculation system that uses only small volumes of bath solution (< 5 ml), reducing the amount of reagents required for long-term experiments lasting many hours. The method employs a custom-made recirculation system involving piezoelectric micropumps and was first used for the acute translational blockade of protein kinase Mζ (PKMζ) synthesis during long-term potentiation (LTP) by Tsokas et al., 2016. In that study, applying antisense-ODN rapidly prevents the synthesis of PKMζ and blocks late-LTP without inducing the compensation by other protein kinase C (PKC) isoforms that occurs in PKCζ/PKMζ knockout mice. In addition, we show that in a low-oxygenation submersion-type chamber, applications of the atypical PKC inhibitor, zeta inhibitory peptide (ZIP) can result in unstable baseline synaptic transmission, but in the high-oxygenation, "Oslo-type" interface electrophysiology chamber, the drug reverses late-LTP without affecting baseline synaptic transmission. This comparison reveals that the interface chamber, but not the submersion chamber, reproduces the effects of ZIP in vivo. Therefore, the protocol combines the ability to acutely block new synthesis of specific proteins for the study of long-term synaptic plasticity, while maintaining properties of synaptic transmission that reproduce in vivo conditions relevant for long-term memory.

Keywords: Long-Term Potentiation (长时程增强), LTP (LTP), Piezoelectric micropump (压电泵), PKMζ (PKMζ), PKM-ζ (PKM-ζ), ZIP (ZIP)

Background

Ex vivo acute brain slices have been a useful experimental model for studies of neural function at the molecular, cellular, and circuit levels. Advances in knockout techniques have also greatly facilitated the generation of genetically modified mice. Hippocampal slices from these mutant mice have become one of the most widely used experimental paradigms for studying both normal synaptic plasticity and its dysfunction in animal models of human psychiatric and neurological disorders.

Several studies, however, have revealed that genetic compensation in response to gene knockout is a widespread phenomenon (for review, see El-Brolosy and Stainier, 2017). Upregulation of related genes following gene knockout can either attenuate or completely compensate for the function of the deleted gene, leading to experimental findings that can be misinterpreted.

Such compensation can be avoided by acutely blocking the de novo synthesis of the target gene product with a brief application of antisense oligodeoxynucleotides (ODNs). ODNs are short single-stranded synthetic DNA molecules, often modified with phosphorothioate linkages to enhance stability. ODNs can bind to complementary regions of a specific mRNA, usually located near the translation start site (AUG, in Figure 1A, top), and physically block the ability of ribosomes to move along the mRNA, preventing synthesis of the protein. Because the effects of ODNs are rapid, the possibility of altered regulation of other genes that can occur with more prolonged gene deletion is reduced. Potential off-target effects may occur, but can be minimized with the appropriate design of the ODN and by pharmacogenetic experiments employing ODNs together with knockouts that lack the target mRNA (Tsokas et al., 2016).

Most previous studies employing ODNs to suppress gene expression in brain slices, however, first infuse the ODN into the brain by intracranial or intraventricular injection, and then soon after the injection prepare the tissue slice (Matthies et al., 1995; Guzowski et al., 2000; Garcia-Osta et al., 2006). Although effective in introducing the ODN into the brain, the injection itself often damages the neural tissue, resulting in suboptimal recordings from brain slices prepared adjacent to the injection site. In addition, the injection delivers the compound as a bolus at a high concentration that then must diffuse through the brain tissue in the intact animal prior to the slice preparation. As a result, the experimenter does not have precise control over the concentration of the compound present in the slice at the time of recording. If a high drug concentration in the bolus injection is required, this in turn can lead to questions about pharmacological specificity.

The method described in this protocol renders the perfusion of brain slices with antisense-ODN as easy as the application of any other soluble reagent or drug. Therefore, the technique may be preferable to the standard method of intracranial or intraventricular injections followed by preparing brain slices. In addition, electrophysiological recordings are in an “Oslo-type” interface brain slice recording chamber. Interface chambers are preferable to submersion chambers for long-term recordings of brain slices because they provide superior oxygenation (for detailed discussion, see Note 1). This protocol was first used in Tsokas et al. (2016) to block specifically the new synthesis of protein kinase Mζ (PKMζ) in response to strong afferent tetanic stimulation (high-frequency stimulation, HFS) without inducing genetic compensation by the other atypical protein kinase C (PKC) isoform, PKCι/λ, as occurs in PKMζ-null mice. Indeed, in these mutant mice, long-term potentiation (LTP) and memory formation appear largely intact because the normal physiological function of PKMζ is largely compensated by PKCι/λ (Tsokas et al., 2016).

Genetic compensation for LTP and spatial long-term memory in PKMζ-null mice was revealed in Tsokas et al. (2016) by a pharmacogenetic analysis of wild-type and PKMζ-null mice using PKMζ-antisense ODN. In these experiments, the normal physiological function of PKMζ was selectively blocked by taking advantage of the specific nucleotide sequence of the PKMζ-mRNA translation start site to design a PKMζ-antisense ODN (Figure 1A) that suppresses the activity-dependent de novo synthesis of PKMζ. Twenty μM PKMζ-antisense was bath-applied to acute wild-type mouse slices and recirculated for 1 h before tetanization and during the critical period of new protein synthesis after tetanization when PKMζ is formed (Osten et al., 1996), i.e., during the temporal window when general protein synthesis inhibitors such as anisomycin are effective in blocking late-LTP induction (Frey and Morris, 1997). The PKMζ-ODN suppressed the new synthesis of PKMζ (Figure 1B) and late-LTP (Figure 1C) without affecting the upregulation of PKCι/λ or the eukaryotic elongation factor 1A (eEF1A), proteins that are also rapidly synthesized in LTP (Tsokas et al., 2016; Figure 1B). Because the turnover of PKMζ is relatively slow (Osten et al., 1996), basal amounts of PKMζ in untetanized slices were unaffected (Tsokas et al., 2016). The application of PKMζ-antisense did not suppress LTP in PKMζ-null mice, in which the target of the PKMζ-antisense is absent (Figure 1C). These results demonstrate that in the mutant mice another molecule compensates for the loss of PKMζ. Conversely, a selective PKCι/λ inhibitor reversed established LTP only in the PKMζ-null mice and not in wild-type mice (Tsokas et al., 2016, Figure 3). This double dissociation between the mechanisms of LTP in PKMζ-null and wild-type mice revealed that when PKMζ is absent there is functional compensation by PKCι/λ.


Figure 1. Acute perfusion of mouse hippocampal slices with PKMζ-antisense ODN (20 μM) via the mp6 micropump system reveals that PKMζ is necessary for late-LTP in wild-type mice, and compensation accounts for late-LTP in PKMζ-null mice. A. Top: Schematic of the PKMζ-antisense. Bottom: Bath pre-application and recirculation of 20 μM biotinylated PKMζ-antisense in ACSF for 1 h in Tg(Thy1-EGFP)MJrs/J mouse hippocampal slices allows highly efficient penetration of the ODN in pyramidal EGFP-filled neurons (green), as verified with cy3-conjugated streptavidin staining of biotinylated ODN (red) post-fixation. B. Bath-applied PKMζ-antisense blocks new synthesis of PKMζ, but not PKCι/λ or the eukaryotic elongation factor 1A (eEF1A) that are also synthesized in LTP. C. Bath-applied PKMζ-antisense blocks late-LTP in wild-type mice but has no effect on LTP in PKMζ-null mice. [A top, B, C: Adapted from Tsokas et al. (2016). For images of Western blots demonstrating the specificity of the PKMζ and PKCι/λ antisera please refer to Figure 2 Supplement 1 and Figure 3 Supplement 2, respectively, from that publication].

The blockade of late-LTP and the suppression of activity-dependent PKMζ synthesis with bath-applied, recirculating PKMζ-antisense was also replicated in acute rat hippocampal slices (See Figures 2A, 2B, and S4B from Hsieh et al., 2017). Taken together, these findings suggest that the crucial pool of PKMζ protein that sustains synaptic potentiation is provided by de novo synthesis in response to tetanization, rather than through the recruitment of pre-existing, basal PKMζ that had been synthesized before the tetanus. Thus, the use of antisense ODN to specifically suppress new synthesis of PKMζ without affecting basal levels of the kinase is advantageous over genetic knockdown/knockout not only because it prevents genetic compensation, but because it distinguishes between basal PKMζ and the pool of PKMζ that is synthesized in response to HFS. The results reveal that only the newly synthesized pool can support late-LTP. A similar argument can be made for the use of acute translational blockade by antisense ODN rather than genetic knockdown/knockout in behavioral experiments designed to elucidate the mechanisms involved in the formation and persistence of long-term memory under physiological conditions in wild-type animals (Tsokas et al., 2016; Hsieh et al., 2017).

Since first described (Tsokas et al., 2016; Hsieh et al., 2017), PKMζ-antisense has been applied acutely on hippocampal slices in two other studies (Dasgupta et al., 2017; Sharma and Sajikumar, 2018) via a peristaltic pump (non-recirculating ACSF). These reports also showed a blockade of LTP.

In our protocol, antisense ODN is delivered by a custom-built recirculation system that perfuses brain slices resting in an “Oslo-type” interface recording chamber with a small (5 ml) recirculating volume of artificial cerebrospinal fluid (ACSF). The system employs the mp6 micropump, a piezoelectric diaphragm pump developed by Bartels Mikrotechnik GmbH (Dortmund, Germany) for the transport of liquids or gases at varying flow rates and/or pressures controlled by an external electronics circuit. For liquids, each mp6 micropump supplies a maximal flow rate of 5.5 ml/min. With parallel connection of multiple mp6 units the volume flows summate. The mp6 can be controlled by the commercially available mp-x controller, or alternatively by the mp6-OEM, both of which are manufactured by Bartels. The mp6-OEM is a small (10.5 x 20.5 x 6 mm) driving circuit capable of generating up to 270 V peak-to-peak voltage at 100 Hz frequency from a 5 V power supply (therefore proper safety measures are required). The OEM controller drives the micropump at adjustable performance and can be integrated into system electronics, a PCB design, or as in the case of this protocol a breadboard.

Each mp6-OEM is intended for operating one mp6 micropump. Therefore, the six mp-x controllers that would be required for driving the mp6 arrays used in this protocol would make the cost of using the commercially available controller quite steep–as opposed to using six mp6-OEMs and building a custom-made circuit at a small fraction of the price. The mp6-OEM has a built-in interface that allows the user to adapt the adjustable parameters (frequency and amplitude) of the rectangular signal generated by the OEM by the use of external components, such as a circuit consisting of a potential divider and a capacitor, or a microcontroller capable of pulse-width modulation (PWM). For the purposes of this protocol, an Arduino Uno is used with a simple program that performs PWM to control the amplitude and/or the frequency. Using an open source Arduino library and a program provided with this protocol, the user may achieve flow rates between 0.2 ml/min and 5.5 ml/min from each mp6 micropump, appropriate for maintaining brain slices (see Figure 20).

The schematic in Figure 2A represents a simplified version of the circuit that controls the flow rate of the pumps which supply ACSF to the recording chamber (inflow micropumps, controlled by the OEM circuit at the bottom via Microcontroller 2). Figure 2A also shows a simplified version of the circuit controlling the suction pumps (outflow micropumps, controlled by the OEM at the top). To achieve laminar flow when the chamber is operated in submersion mode, i.e., during ODN delivery prior to stimulation and recording, slices are perfused on two sides–at the top and the bottom of the mesh. Two micropumps driven by two separate OEMs are therefore used for controlling the inflow: one pump perfuses the top of the mesh, and the other supplies ACSF to the part of the chamber below the mesh (Figures 15A and 15B). A “sandwich” consisting of four mp6 pumps connected in parallel fashion is used to supply the suction that completes the ACSF recirculation circuit (Figure 15C). These four mp6 micropumps operate constantly at maximal flow rate and are therefore controlled by a simpler version of the circuit used for the two inflow micropumps, which does not perform pulse-width modulation.

A separate Arduino Uno microcontroller (Microcontroller 1) is used to turn all the mp6 micropumps on/off in a concerted fashion according to inputs received by the ADC and the software driving the electrophysiology experiment. In this manner, the recirculation system can be used in combination with a peristaltic pump via a three-way valve and activated only during drug application.

The first part of this protocol is a description of how to build the electronic circuit that controls the operation and flow rate of the mp6 micropump arrays. The second part provides a detailed hippocampal slice preparation protocol for electrophysiology in interface chambers. The protocol includes modifications of the classic method described in the scientific literature, which, based on our experience, we believe are necessary for making high-quality slices suitable for long electrophysiological recordings and subsequent biochemical and immunocytochemical analysis.

In addition, we provide evidence for the advantage of interface over submersion electrophysiology chambers for long recordings of acute brain slices required for the study of long-term synaptic plasticity (see Note 1). Whereas low-oxygen submersion chambers show unstable baseline synaptic transmission following long-term applications of the zeta inhibitory peptide (ZIP), the interface electrophysiology chambers maintain stable baseline synaptic transmission, as observed after applications of ZIP in vivo (Pastalkova et al., 2006). Thus, because brain slices maintained in interface chambers more closely preserve the in vivo physiological state, they are the preferred method for investigating the long-term synaptic plasticity thought to underlie learning and memory.


Figure 2. Color-coded schematic of the micropump control circuit and photographs of the micropump Molex connector. A. The cables connecting the different components are color-coded for easy assembly of the circuit; a step-by-step outline is provided in Figures 4-14. B. Color-coded scheme of the connections on the Molex connector. The connections labeled -P1, +P1, +P2, and -P2 should be connected to the four output pins of the OEMs as dictated by the colors. For fast and secure fastening/unfastening of the micopump assemblies, use a Pi Cobbler Breakout and associated cable. C. Photograph of an mp6 micropump and Molex connector from B. Note that only one side of the micropump has markings (the company logo and micropump serial number). Slide the micropump through the slot, in the orientation shown, and close the Molex component to secure the connection.

Materials and Reagents

  1. Double-sided adhesive tape (Amazon, model: QK-8285B0134CUFU0_loc)
  2. Single Edge Razor Blade (GEM Personna, Electron Microscopy Sciences, catalog number: 71972)
  3. Double Edge Razor Blade (Electron Microscopy Sciences, catalog number: 71998)
  4. Pyrex Graduated Cylinder 2,000 ml (Corning, VWR, catalog number: 3022-2L)
  5. Pyrex Graduated Cylinder 1,000 ml (Corning, VWR, catalog number: 3022-1L)
  6. Pyrex Graduated Cylinder 100 ml (Corning, VWR, catalog number: 70075-100)
  7. Pyrex Culture Petri Dish 90 mm (Corning, VWR, catalog number: 7740)
  8. Pyrex Media Storage Bottle, 1 L (Corning, VWR, catalog number: 1395-1L)
  9. Pyrex Gas-Dispersion Tube (Corning, VWR, catalog number: 39533-12C)
  10. Pyrex Graduated Beaker, 250 ml (Corning, VWR, catalog number: 1000-250)
  11. Pyrex Graduated Beaker, 50 ml (Corning, VWR, catalog number: 1000-50)
  12. Pyrex Graduated Beaker, 20 ml (Corning, VWR, catalog number: 1000-20)
  13. VWR Erlenmeyer Flask, 250 ml Erlemeyer flask (VWR, catalog number: 75804-646)
  14. 15 ml conical tube (VWR, catalog number: 62406-200)
  15. Scalpel Handle #3 (Fine Science Tools, catalog number: 10003-12)
  16. Scalpel Blade #10 (Fine Science Tools, catalog number: 10010-00)
  17. Surgical Scissors-ToughCut (Fine Science Tools, catalog number: 14054-13)
  18. Extra Fine Bonn Scissors (Fine Science Tools, catalog number: 14084-08)
  19. Friedman-Pearson Rongeurs (Fine Science Tools, catalog number: 16221-14)
  20. Spatula (VWR, catalog number: 82027-490)
  21. Ellipso-spoon Spatula ((VWR, catalog number: 100493-952)
  22. Camel Hair Brush #1 (Ted Pella, Inc., catalog number: 11859)
  23. Insect pin (Fine Science Tools, catalog number: 26000-50 )
  24. Whatman Grade 5 Filter Paper 55 mm (Whatman, VWR, catalog number: 1005 055)
  25. Whatman Grade 5 Filter Paper 90 mm (Whatman, VWR, catalog number: 1005 090)
  26. Transfer Pipets (Fisherbrand, Fisher Scientific, catalog number: 13-711-7M)
  27. Tubing Extension with 1.3 mm I.D. (Cole Parmer, catalog number: SK-95809-32)
  28. Tygon Tubing, 1.6 mm I.D. (Cole Parmer, catalog number: SK-06407-33)
  29. Teflon/PTFE Tubing, 1.6 mm I.D. (Cole Parmer, catalog number: SK-95231-00)
  30. Three-way valves (Cole Parmer, catalog number: SK-30600-02)
  31. Female and male gold-plated Pins (Fine Science Tools, catalog numbers: 19003-01 and 19-003-00)
  32. Calcium Chloride Solution, 1 M (CaCl2) (Teknova, catalog number: C0477, room temperature)
  33. Magnesium Chloride Solution, 1 M (MgCl2) (Teknova, catalog number: M0304, room temperature)
  34. Magnesium Sulfate Solution, 1 M (MgSO4) (Teknova, catalog number: M3003, room temperature)
  35. Potassium Chloride (KCl) (Fisher Scientific, catalog number: P9541-1KG, room temperature)
  36. Sodium Bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S5761-500G, room temperature)
  37. Sodium Chloride (NaCl) (Sigma-Aldrich, catalog number: S271-3, room temperature)
  38. Sodium Phosphate Monobasic (NaH2PO4) (Sigma-Aldrich, catalog number: S5011-500G, room temperature)
  39. D-(+)-Glucose (Sigma-Aldrich, catalog number: G7528-1KG, room temperature)
  40. Isothesia (Isoflurane) Solution (C102ClF5O) (Henry Schein Animal Health, catalog number: 029404, room temperature)
  41. Antisense and Scrambled ODN (reverse phase cartridge-purified) with phosphorothioate linkages 5’-3’ on three terminal bases at each end to protect against nuclease degradation, as per Tsokas et al. (2016), Hsieh et al. (2017) (Gene Link, Hawthorne, NY, 4 °C)
  42. 100% Oxygen (TW Smith)
  43. 95% Oxygen-5% Carbon Dioxide (TW Smith)
  44. 10x Stock Dissection ACSF Buffer (see Recipes)
  45. 10x Stock Recording ACSF Buffer (see Recipes)
  46. 1x Dissection ACSF Buffer (see Recipes)
  47. 1x Recording ACSF Buffer (see Recipes)

Equipment

  1. 4 °C cold room
  2. -70 °C freezer: Thermo Scientific Forma 88000 Series -86 °C (Thermo Scientific, model: 88600D) 
  3. Water bath: Fisherbrand Isotemp Digital-Control Water Bath Model 202 (Fisher Scientific, model: 15-462-2Q)
  4. Corning Round Ice Bucket with Lid, 4 L (Fisher Scientific, catalog number: 07-210-123)
  5. “Oslo-type” Interface Brain Slice Recording Chamber (Fine Science Tools, catalog number: 21000-02)
  6. Warner TC-344C Dual Channel Temperature Controller (Warner Instruments, catalog number: 64-2401)
  7. Dissection Binocular Stereo Zoom Microscope with Boom Stand (Nikon, Morrell Instrument Co., catalog number: SMZ800N)
  8. Fiber Optic Light Source (Dolan-Jenner MILEDUSB1 MI-LED-US-B1)
  9. Dual Gooseneck Fiber Optic Assembly (Dolan-Jenner EEG3736 002144113036)
  10. Neuroprobe Amplifier, Model 1600 (A-M Systems, catalog number: 680100)
  11. Model 1600 Headstage (A-M Systems, catalog number: 681500)
  12. ADC Connector Box (National Instruments, catalog number: BNC-2090A)
  13. ADC PCI Bus Card (National Instruments, catalog number: PCIe-6323)
  14. Dell Optiplex 3040 (Amazon, catalog number: B01E8QRSAG)
  15. ISO-flex Stimulator (AMPI, catalog number: ISO-flex)
  16. Narishige MX-2 Micromanipulator
  17. Narishige MM-3 Micromanipulators
  18. Flaming/Brown Pipet Puller (Sutter Instruments, catalog number: P-97)
  19. McIlwain Tissue Chopper (McIlwain Lab, Ted Pella, Inc., catalog number: 10180)
  20. Plastic Disc (Ted Pella, Inc., catalog number: 10180-01)
  21. Rodent Guillotine (WPI, World Precision Instruments, catalog number: DCAP)
  22. Isoflurane Vaporizer (VSS, catalog number: 300 Series)
  23. Anesthesia Induction Chamber (Leica Biosystems, catalog number: 39481051/3)
  24. Six mp6-OEM controllers (Mp6-oem, Bartels Mikrotechnik GmbH, Servoflo catalog number: Mp6-oem)
  25. Six mp6 micropumps (mp6, Bartels Mikrotechnik GmbH, Servoflo catalog number: mp6)
  26. Six Passive Check Valves mp-cv (Bartels Mikrotechnik, Servoflo, catalog number: mp-cv)
  27. Six FCC connectors (Molex; Digi-Key, catalog number: 0039532045)
  28. Two Arduino Uno Microcontrollers (Adafruit Industries, catalog number: 782-A000066)
  29. Two 8-channel Bi-directional Logic Level Converters (Adafruit Industries, catalog number: TXB0108)
  30. Assembled Pi Cobbler Breakout Board + 26-pin Ribbon Cable for Raspberry Pi Models A & B (Adafruit, catalog number: 914)
  31. Shield stacking headers for Arduino (R3 Compatible) (Adafruit, catalog number: 85)
  32. Male/male Jumper Wires (Adafruit, catalog numbers: 758, 1956)
  33. Jumper Wire Kit–pre-cut and bent in right angles, 700 pcs (Sparkfun Electronics, catalog number: PRT-14671)
  34. MC001 Alpha Wire Multi-Conductor Cables (Mouser, catalog number: 602-1102-100)
  35. WBU-206 Breadboard 31 mm x 175 mm x 230 mm (Newark Electronics, catalog number: 18M7115); Alternative: Digilent 340-002-1 (Digi-Key, catalog number 1286-1220-ND)
  36. 10K-Ohm 15-Turn Cermet Potentiometer/Trimmer (RadioShack, catalog number: 2710343)
  37. RS Pro 1440976, AC to DC Desktop Power Supply, 5 VDC, 7.5 A (Allied Electronics, catalog number: 71259700)
  38. SPST Push On/Off Switch (RadioShack, catalog number: 2750011)
  39. Six Y-shape tubing connectors, barbed (Servoflo, catalog number: mp-y)
  40. Heat Shrink Tubing (Mouser, catalog number: 650-VER332SP)
  41. Weller WTCPT Soldering Station (Digi-Key, catalog number: WTCPT-ND)
  42. Solder (Digi-Key, catalog number: KE1106-ND)
  43. Heat gun: AlphaWire Fit Gun-1 (Digi-Key, catalog number: FGUN1NC032-ND)
  44. Multimeter Fluke 177 (Digi-Key, catalog number: 614-1020-ND)
  45. USB cables (Amazon, ASIN: B003ZZH2U8)
  46. Nine custom-made plexiglass spacers (MSC Industrial Supply, catalog number: 63388953)
  47. Six custom-made Cork pad spacers (Cork Pad sheet from Amazon, ASIN: B001ACMBO0)
  48. Flathead 2-56 ¾ inch screws: (MSC Industrial Supply, catalog number: 40914715)
  49. Copper 33 Gage, 0.011 Inch Wire Diameter, 16 x 16 Mesh (MSC Industrial Supply, catalog number: 52427291)
  50. Velcro 1" Wide Adhesive Backed Hook & Loop Roll (MSC Industrial Supply, catalog number: 67127480)
  51. Clamps (Fisher Scientific, 05-769-7Q; S13743; 02-215-466; 02-300-206)
  52. Optional: Dissolved Oxygen Kit (Atlas Scientific, Atlas Scientific, catalog number: KIT-106)

Software

  1. winLTP (WinLTP Ltd. and The University of Bristol, https://www.winltp.com)
  2. Arduino Software (IDE) (Arduino, https://www.arduino.cc/en/Main/Software)
  3. Atlas Scientific Dissolved Oxygen OEM sample code (https://www.atlas-scientific.com/_files/code/oem-do-sample-code.pdf)

Procedure

Part I: ACSF Recirculation System Assembly

  1. Assembly of the Micropump Control Circuit
    For a simplified schematic with color-coded cables refer to Figure 2A. For detailed, step-by step photos of the assembly of the circuit, consult Figures 4-14. For a table listing all the connections required for the mp6 micropump control circuit, see Figure 3.


    Figure 3. Table listing all the breadboard connections required for the mp6 micropump control circuit


    Figure 4. Steps 1-4 of the assembly of the mp6 micropump control circuit. See text for details.

    1. To fit all the necessary components, a breadboard with 3 terminal strips and 63 rows of 10 tie points per terminal strip is required (Figure 4). Such breadboards include the Digilent 340-002-1 or the Wisher WBU-206. Each of these has 5 power distribution buses and 3 terminal strips. Remove (unscrew) the rightmost distribution bus and the adjacent terminal strip.
    2. Attach with screws or Velcro pads the two Arduinos–ideally in a staggered fashion to allow easy access to the two USB ports, as shown in Figure 4.
    3. Connect the 5 Volt DC Power Supply to the horizontal distribution bus of the breadboard (Figure 4, designated by the red “plus” and the black “minus” signs). Include a simple toggle SPST push button “master switch” (not shown), for switching the power supply to the circuit.
    4. Connect the power and ground of all vertical distribution buses to 5 V and ground, respectively, of the horizontal distribution bus (Figure 4)


      Figure 5. Steps 5-6 of the assembly of the mp6 micropump control circuit. See text for details.

    5. On the middle (now rightmost) terminal strip attach the two 10K-Ohm potentiometers, and connect them to power and ground, as shown in Figure 5.
      Note: The screws that rotate the wiper and vary the resistance in each potentiometer should be facing away from each other, therefore allowing enough clearance for the mini screwdriver that will be used to make adjustments in Step 6.
    6. Using a mini screwdriver, adjust voltage to get 1.3 V across the wiper of the top potentiometer and 1.8 V across the bottom.
      Note: For safety, you should disconnect the power supply when proceeding with the next steps of the protocol.


      Figure 6. Steps 7-8 of the assembly of the mp6 micropump control circuit. See text for details.

    7. On the terminal strip that includes the two potentiometers also install the two 8-channel logic level converters (Figure 6). Connect VCCB on each logic level converter to 5 V and GND to ground (Figure 6; also refer to Figure 2A for pinout diagram and voltage information).
    8. Connect VCCA and OE on the top logic level converter to 1.3 V and on the bottom converter to 1.8 V (see gray jumper wires in Figures 6; also refer to Figure 2A for voltage and pin information).
    9. Attach the 26-pin Assembled Pi Cobbler Breakout Board. Use the shield stacking headers to elevate the Breakout Board, and therefore allow enough clearance for the third and fourth mp6-OEM controllers that will be attached next (Step 10) and are directly adjacent to the Breakout Board (see Figure 15D).


      Figure 7. Steps 9-14 of the assembly of the mp6 micropump control circuit. See text for details.

    10. Connect the six mp6-OEM controllers to the leftmost terminal strip of the breadboard as shown in Figure 7 (also refer to Figure 2A).
      Note: The bottom two mp6-OEM controllers will supply the inflow lines (perfusing the top and bottom sides of the mesh) of the bath of the interface chamber, and their flow rate is adjustable by the bottom Arduino Uno, which is connected to Pin 2 (Cyan, added in Step 22) (Figure 2, and Figures 15A and 15B). The top four OEMs are connected to the four-pump array that supplies the suction (Figure 15C), and their flow rate is constant (set at maximal). For these OEMs, Pin 2 and Pin 3 are shorted and do not receive any input from the bottom Arduino Uno (Figure 2A). Also notice that in all six of the OEMs Pin 5 and Pin 10 are unused.
    11. Short Pins 11 and 12 on all six mp6-OEMs, using U-shaped loops made out of cut jumper wires, long enough to connect two contacts on adjacent breadboard rows (see six-pointed asterisks in Figure 7; refer to Figure 2A for numbering of pins).
    12. Similarly, short Pins 2 and 3 of the top four OEMs (see five-pointed asterisks in Figure 7; refer to Figure 2A for numbering of pins).
    13. Connect the ground (Black) to Pin 1, marked with a white spot of all six mp6-OEMs (Figure 7; refer to Figure 2A for numbering of pins).
    14. Connect 5 V (Red) to Pin 14 of all six mp6-OEMs, as shown in Figure 7 (refer to Figure 2A for numbering of pins).


      Figure 8. Step 15 of the assembly of the mp6 micropump control circuit. See text for details.

    15. Connect 1.3 V (Yellow), from A1, A2, A3, A4, A5, A6 of the top logic level converter, to Pin 4 of all six mp6-OEMs (Figure 8; only two such connections are shown in the schematic of Figure 2A).


      Figure 9. Step 16 of the assembly of the mp6 micropump control circuit. See text for details.

    16. Connect 1.8 V (Orange), from A1, A2, A3, A4, A5, A6 of the bottom logic level converter, to Pin 13 of all six mp6-OEMs. (Figure 9; only two such connections are shown in the schematic of Figure 2A).


      Figure 10. Steps 17-18 of the assembly of the mp6 micropump control circuit. See text for details.

    17. Connect Pin 6 (Brown) to Pins Left 1, 3, 5, 7, 9, 11 of the Assembled Pi Cobbler Breakout (Figure 10).
    18. Connect Pin 7 (Green) to Pins Left 2, 4, 6, 8, 10, 12 of the Assembled Pi Cobbler Breakout (Figure 10).


      Figure 11. Steps 19-20 of the assembly of the mp6 micropump control circuit. See text for details.

    19. Connect Pin 8 (Blue) to Pins Right 2, 4, 6, 8, 10, 12 of the Assembled Pi Cobbler Breakout (Figure 11).
    20. Connect Pin 9 (Purple) to Pins Left 1, 3, 5, 7, 9, 11 of the Assembled Pi Cobbler Breakout (Figure 11).


      Figure 12. Step 21 of the assembly of the mp6 micropump control circuit. See text for details.

    21. Connect Inputs B1, B2, B3, B4, B5, B6 of the top logic level converter to Digital Pins 2, 3, 4, 5, 6, 7, respectively, of the top Arduino Uno, as shown in Figure 12 (White cables).


      Figure 13. Step 22 of the assembly of the mp6 micropump control circuit. See text for details.

    22. Connect Inputs B1, B2, B3, B4, B5, B6 of the bottom logic level converter to Digital Pins 8, 9, 10, 11, 12, 13, respectively, of the top Arduino Uno, as shown in Figure 13 (Magenta cables).


      Figure 14. Step 23 and complete overview of the assembly of the mp6 micropump control circuit. See text for details.

    23. Connect Outputs 9 and 10 (PWM0 and PWM1) of the bottom Arduino Uno to Pin 2 (Cyan) of the bottom two OEMs (Figure 14).
    24. Connect one of the Digital outputs of the ADC to TX 1 and GDN of the top Arduino Uno (Figure 2A). This connection will turn on and off the recirculation perfusion controlled by the mp6 pumps, as directed by the winLTP Program.
    25. Connect the two Arduino Uno units to the computer, using USB connection. 
    26. Connect the GDN of the bottom Arduino to the central ground of the electrophysiology rig. (Figure 2A. The top Arduino is grounded via the ADC ground—see Step 24).
    27. Copy and load into the top Arduino controlling all six micropumps (Microcontroller 1) the program shown in Figure 18.
    28. Copy and load into the bottom Arduino controlling the two inflow micropumps (Microcontroller 2) the program shown in Figure 19.

  2. Assembly of the mp6 micropump array (see reference 29)
    1. Assemble the six cables connecting the six Molex connectors (one per mp6 micropump) to the Breakout Board 26-pin ribbon cable, as shown in Figures 2B and 15E. The color scheme of Figures 2B and 2C serves as a guide. Use four male gold-plated pins and a 32AWG 4-wire multi-conductor cable (e.g., the MC001 Alpha Wire Multi-Conductor Cable) to connect the four pins of the Molex connector to the ribbon cable. Each Molex connector and associated multicolor cable will be inserted into four sockets of the ribbon cable (wire arrangement, clockwise from top left socket: brown, purple, blue, green; repeat for all six Molex connectors). Only 24 (6 x 4) out of the 26 sockets of the ribbon cable will be used.
      Note: Disregard the colors of the Alpha Wire multicolor cables in Figures 15A, 15B, 15C, 15E and 15F, which are unrelated to the color scheme of Figure 2B.
    2. Position both components as indicated in Figure 2C, the mp6/mp6-pp facing upwards (with its company logo and serial number markings visible from above), and the Molex connector with the four small openings facing down. Then slide the mp6/mp6-pp flex into the Molex connector. Clamp the Molex connector to complete the connection between both components.
    3. Manufacture three custom-made plexiglass spacers similar to the ones shown in Figures 15A and 15B. Also prepare cork pads cut to size in order to compensate for differences in the thickness of the micropump and the Molex connectors.
    4. Make an assembly consisting of two mp6 micropumps as shown in Figures 15A (front) and 15B (side) as follows: First, glue the marked side of micropump 1 (mp6-1) on the plexiglass spacer using double-sided adhesive tape. Glue cork pads to the unmarked side of the first micropump. The order of parts in the resulting component is: plexiglass/mp6-1/cork.
    5. Repeat these steps for micropump 2 (mp6-2), but in the opposite order, i.e., with the cork pad glued to marked side and the plexiglass spacer on the unmarked. The order of parts in the resulting component is: cork/mp6-2/plexiglass.
    6. Build a four mp6 micropump assembly (Figure 15C) consisting of the two plexiglass spacer/mp6/cork pad components, by placing the third (unglued) plexiglass spacer in between, so that order of parts in the completed array is:
      plexiglass/mp6-1/cork/plexiglass/cork/mp6-2/plexiglass.
    7. The resulting assembly can be held together either with pieces of double-sided adhesive tape, or by a clamp, or with long screws that pass through drilled holes into the plexiglass spacers. In the latter case, make sure to tap threads into the holes of only one out of the three spacers.
    8. Make mini-Faraday cages out of copper wire mesh (Figure 15F) to allow the elimination of the noise generated by the piezoelectric micropumps (see Note 3). Ground the mini Faraday cages on the main ground of the airtable.
    9. The completed assemblies consist of either two or four mp6 micropumps (Figure 15F). As discussed above, four pumps connected in parallel are required for the suction, and two pumps with independent inlets and outlets perfuse the top and bottom of the hippocampal slice, from both sides of the mesh in the recording chamber.


      Figure 15. Different views of the assembled micropump arrays and associated components. A and B. Two mp6 micropumps are stacked in parallel connection, which causes their volume flows to summate. Each such “sandwich” array consists of three custom-made plexiglass panels, one of which is threaded so that screws can hold the assembly together. Cork pads cut to size allow for precise contact of different surfaces. C. Two assemblies shown in (A) and (B) stacked on top of each other, constitute the pump array that controls the suction. Notice the y connectors and mp-cv valves. D. The 26-pin Assembled Pi Cobbler Breakout Board is connected to 26-pin shield stacking headers with longer pins, so as to allow enough clearance for two of the mp6-OEM controllers that are attached directly adjacent to it. E. Cable that connects six micropumps to the Pi Cobbler Breakout Board. F. Mini-Faraday cages made of copper wire mesh to allow grounding the noise from the pumps.
      Note: Alpha Wire multiconductor cables have their own color scheme which is unrelated to the color scheme of Figures 2B, 2C, 9 and 11.

    Part II: Rodent Hippocampal Slice Preparation

    1. Prepare Dissection and Recording Solutions: Prepare two stock 10x ACSF solutions, one for dissection (Dissection 10x ACSF) and one for recording (Recording 10x ACSF). Maintain the two stock 10x ACSF solutions at 4 °C until the day of the experiment.
    2. On the day of the experiment, prepare 1x Dissection ACSF and 1x Recording ACSF.
    3. Oxygenate both solutions for at least 20 min at room temperature with 95% O2/5% CO2.
    4. Reducing the temperature of the slice helps prevent ischemic damage (see Note 2). To rapidly chill room-temperature 1x Dissection ACSF Buffer to an appropriate cold temperature without freezing the buffer, place it into a -70 °C freezer for 45 min. If ice crystals form, remove them with a small spoon or sieve to avoid contact with, and possible damage to the brain tissue. After removing from the freezer continue to oxygenate the Dissection ACSF with 95% O2/5% CO2 at 4 °C.
    5. Prepare the interface electrophysiology rig: Heat the water jacket of the interface chamber so that the temperature of the recording chamber is 31.5 °C. Preheat 250-500 ml of the 1x Recording ACSF by placing it in a 32 °C water bath while oxygenating with 95% O2/5% CO2. The purpose of preheating the solution to a temperature slightly above the temperature of the recording chamber is to prevent the formation of bubbles in the tubes and under the mesh when the solution is heated again after passing from the heat jacket. Start perfusing the bath with 1x Recording ACSF, at a flow rate of 500 μl/min.
    6. Deeply anesthetize the animal in the anesthesia chamber, using an Isoflurane vaporizer: Apply 100% O2 for 2 min, followed by 100% O2 + 1.5% Isoflurane for 1 min, followed by 100% O2 + 2.5% Isoflurane for 2 min.
    7. When the animal is deeply anesthetized, decapitate it with a small animal guillotine. Submerge the decapitated head inside a metal surgical tray sitting on ice in an icebox and filled with oxygenated ice-cold 1x Dissection ACSF.
      Note: Following the decapitation of the animal, the preparation of the brain slices should be completed within less than 10 min.
    8. Quickly remove the brain: expose the skull by making an incision with a single edge razor blade on the scalp.
    9. Using a pair of surgical scissors with their sharp pointed blade working along the inner side of the skull, cut along the sagittal suture from the foramen magnum to the forehead.
    10. Using the surgical scissors, make one cut at the foramen magnum on each temporal side of the skull, then cut across the frontal bone along the coronal suture. Carefully pry the skull open with a rongeur and expose the brain.
    11. Holding the skull upside down, sever the cranial nerves that hold the brain to the skull using a spatula and allow the brain to fall into a 50 ml beaker containing ice-cold oxygenated 1x Dissection ACSF.
    12. Using an icebox, transfer the beaker with the brain to a 4 °C cold room.
    13. Isolate the hippocampus: use a 90 mm Pyrex Culture Petri Dish covered with a moistened filter paper as a dissecting platform.
    14. Place the chilled brain on the platform and bisect it along the longitudinal fissure. Place the right hemibrain in a 50 ml beaker containing ice-cold oxygenated 1x Dissection ACSF.
    15. To isolate the hippocampus of the left hemibrain, sever what remains of the midbrain and brain-stem using a flat spatula. Scoop out the thalamus to expose the ventral face of the hippocampus nested inside the cortex and clearly visible as a curved structure. Sever the septal and temporal connections of the hippocampus from the cortex with a spatula. Using a filled plastic Pasteur pipet, gently squeeze a few droplets of ice-cold oxygenated 1x Dissection ACSF into the pocket of the lateral ventricle to distinguish the border of the fimbria.
    16. Gently insert into the lateral ventricle a fine brush moistened with ice-cold oxygenated 1x Dissection ACSF and roll the hippocampus out gently from the surrounding cortex. Isolate the peeled-out hippocampus by cutting it free from the cortex using a flat spatula. Place the left hippocampus in a 20 ml beaker with ice-cold oxygenated 1x Dissection ACSF.
    17. Repeat previous steps with right hippocampus.
    18. Slicing: The slicing stage should be prepared in advance and can be reused multiple times. It consists of a circular thin piece of cork pad of equal diameter to the plastic disc of the McIlwain tissue chopper and glued on it with parallel pieces of double-sided adhesive tape. The clamps of the McIlwain tissue chopper dissection platform can be adjusted to match the thickness of the slicing stage (Figure 16C).
    19. Place a piece of filter paper on the slicing stage and moisten it with chilled oxygenated 1x Dissection ACSF.
    20. Using a fine brush (or a spatula with its flat side bent at right angles) gently lift the hippocampus from the bottom of the beaker.

       
      Figure 16. Photographs showing proper positioning of the left rat hippocampus on the McIlwain tissue chopper dissection platform (refer to text for details)

    21. Lay the isolated hippocampus on the slicing stage with its top side up (the dorsal face containing the alveus). It is often possible to view striations on the alvear surface of this side, with oblique light from a fiber optic. Using the brush gently straighten the bottom surface of the hippocampus so that it lies completely flush on the moistened filter paper.
    22. Only the dorsal hippocampus will be sliced from each side. To immobilize the hippocampus, one may therefore affix the ventral hippocampus (the thicker end) on the cork of the slicing stage with an insect pin (Figure 16A).
    23. Slide the slicing stage with the affixed hippocampus through the clamps of the McIlwain tissue chopper dissection platform (Figure 16C).
    24. Rotate the slicing stage on the platform so that the dorsal hippocampus is properly oriented with respect to the razor blade: the blade should make a 15° angle with the transverse axis of the dorsal hippocampus (Figures 16B, 16C). Excitatory pathways are better preserved when an angle of 15 to 30° from this axis is used (Alger et al., 1984). In general, the alvear striations will tend to run parallel to the blade at this orientation. If the hippocampus on the left hemibrain is used, it will look like an upright “U” as viewed from above with the dorsal hippocampus on the right and the (pinned) ventral hippocampus on the left (Figures 16A-16C). To achieve the proper cutting angle, rotate the slicing stage 15° counterclockwise. Similarly, if the right hippocampus is used, it will resemble an inverted “U”, and the stage is rotated 15° clockwise.
    25. Wet the blade with ACSF and make the slices by lowering the blade through the hippocampus. Each slice should stick on the wet surface of the blade as it ascends. The first 3 (most dorsal) complete slices are usually discarded. Depending on the age of the animal, 6 to 8 slices of 450 mm thickness can be obtained from rats (Figure 16B) and 4-5 such slices from mice. The speed and the strength of impact of the blade should be adjusted so that the slices are cut gently, but quickly enough to avoid sticking of the uncut hippocampus on the ascending blade. The blade should not slam on the surface of the cutting stage; ideally it should just dimple the surface of the wet filter paper on the chopping stage.
    26. Quickly remove the slices from the blade as they are produced using a moistened fine sable brush. Immediately after making each slice, transfer it to a test tube filled with ice-cold oxygenated 1x Dissection ACSF. Take great care to avoid stretching or excessively bending the slices during handling. One method is to pick the slices from the blade with the brush premoistened with ice-cold oxygenated 1x Dissection ACSF, using a gentle rolling motion. Some of the ACSF moisture on the brush will also stick on the blade and help the next slice stick to the blade after it has been sliced off the hippocampus.
    27. Immediately after making the slices, transfer them to the interface recording chamber to recover for at least 2 h. The slices should rest on a mesh at the gas-liquid interface, forming a small meniscus around them (Figures 17A, 17B and 17C), and should be constantly superfused from both their top and bottom side with (non-recirculating) 1x Recording ACSF, at a flow rate of 500 μl/min.

    Note: Please also consult the following video articles offering detailed information on acute hippocampal slice preparation for long-term recordings: Villers and Ris (2013); Shetty et al. (2015).


    Figure 17. Photographs showing proper positioning of a rat hippocampal slice in an Oslo-type interface recording chamber. A. The slice lies on a mesh, near the wall of a recording bath. The chamber has two such recording baths, both of which are covered by a removable convex canopy that creates a humidified, oxygen-rich atmosphere above the slice. The canopy has a triangular opening that allows recording from each slice individually (while the slice in the other bath remains covered). Each slice is constantly superfused with ACSF flowing into the bath through an inflow tube and out through an outflow conduit (across from the inflow), which leads to the suction well (the suction is provided by a yellow syringe needle). B and C. At interface, a small meniscus is formed around the slice. A larger meniscus is formed around the walls of the bath.

    Part III: Application of oligodeoxynucleotides to slices in recirculation mode

    In ODN experiments, after recovery from dissection in interface mode, increase the bath level to fully submerge the slices and allow the superfusate containing 20 μM of the oligodeoxynucleotide to recirculate (5 ml total volume at 5 ml/min for 30 min). To achieve this, follow these steps:

    1. Copy and load the program of Figure 18 on the top Arduino of Figure 14 (“Microcontroller 1” in Figure 2A). Change the value of “delay” to “1800000”, save the new program and reload to top Arduino. Upon activation by the digital output of ADC via the winLTP program, the top Arduino will turn on all six of the micropumps for 30 min.
    2. Load the Arduino PWM library (see link) and copy and load the program of Figure 19 on the bottom Arduino of Figure 14 (“Microcontroller 2” in Figure 2). Change the “int32_t clockfrequency” value to “200”, save the new program and reload to the bottom Arduino. Upon activation by the digital output of the ADC via the winLTP program, the bottom two pumps will supply a flow rate of 5 mL/min (Figure 20) to the inlets of the recording chamber (the four ouflow pumps of the suction, once on, will always operate at maximum flow rate).
    3. Turn off the peristaltic pump, and at the same time turn off the vacuum suction.


      Figure 18. Arduino program that activates simultaneously all six mp6 oems and keeps them active for a designated period. The program allows turning all six of the mp6 micropumps on simultaneously when prompted by one of the digital outputs of the ADC, which is in turn controlled by the “Slow0 Perfusion Change” function of the winLTP program. By changing the “delay” value (highlighted in yellow) the slices can be perfused for variable intervals. In this particular example the pumps will remain on for 1800000 ms (i.e., 30 min).

    4. Turn on the piezoelectric pump circuit using the SPST push button master switch. 
    5. Use a three-way valve to manually switch between the peristaltic pump drawing solution from a main reservoir (typically a 250 ml Erlenmeyer flask held inside a water bath with a clamp) and the two-micropump assembly of the bath inlet, which should be drawing solution from a 15 ml conical tube containing the recirculating solution with the ODN (inside the same water bath as the main reservoir).
    6. Start the winLTP program with Sequential Protocol Scripting and Automated Perfusion Control (Anderson et al., 2012; see also Chapter 10 of the WinLTP software manual (Anderson, 2018).
      Note: The automated perfusion control is currently only supported by National Instruments M- or X-Series ADC boards). Use the Slow0 Perfusion Change for controlling one perfusion line to one extracellular slice chamber.


      Figure 19. Arduino program that performs pulse width modulation (PWM). The program allows changing the frequency of the signal generated by the mp6-OEM from the default value, to reduce the mp6 micropump flow volume from its maximum value (5.5 ml/min). The frequency of the OEM signal is altered by changing the value of “int32_t clockfrequency” (highlighted in yellow). The current value “int32_t clockfrequency = 75” yields a flow rate of approximately 2.5 ml/min (see Figure 20).
      Note: Library download link: https://code.google.com/archive/p/arduino-pwm-frequency-library/downloads.

    Note: The digital output from the National Instruments M- or X-Series board is connected to Arduino 1, and it will activate both the assembly consisting of the two independent micropumps that control the inlets to the bath, as well as the multipump array that controls the suction. Make sure the tube at the output of the aspirator leads the solution back to the 15 ml conical tube to complete the recirculation circuit.

    1.  If necessary, adjust the height of the suction tube in the suction well of the chamber to raise the bath level and fully submerge the slice.
    2. After 30 min of recirculation in the submersion mode and to begin recording, lower the bath level of the ODN-containing solution by lowering the aspirator tube to interface level and return the flow rate to 0.5 ml/min for the remainder of the experiment by changing the “int32_t clockfrequency” value from 200 to 10, and loading again the program of Figure 19 to the Bottom Arduino Uno.


      Figure 20. Changing the flow rate of single mp6 micropump by pulse-width modulation (PWM). By inserting different values for “int32_t clockfrequency” (x-axis of the graph), the flow rate (y-axis) can be changed from the low rates of a few hundred microliters per min (shown in the insert) required for interface chamber recordings, to a maximal flow rate of 5.5 ml/min.

    3. Place stimulating and recording electrodes in the hippocampal layers of interest and begin electrophysiological recordings.
    4. For experiments to study long-term synaptic plasticity lasting many hours, record field EPSPs (fEPSPs) with a glass extracellular recording electrode (2-5 MΩ) placed in the CA1 stratum radiatum, with concentric bipolar stimulating electrodes placed on either side within CA3 or CA1. Exclude from study slices if initial analysis shows fEPSP spike threshold < 2 mV. Confirm independence between the two stimulated pathways by the absence of paired-pulse facilitation between the two pathways. Set the baseline fEPSP at 25% of the spike threshold and monitor it by delivering stimuli at 0.033 Hz to each pathway. Induce LTP by strong HFS, consisting, for example, of standard two 100 Hz-1s tetanic trains, spaced 20 s apart, which is optimized to produce a relatively rapid onset of protein synthesis-dependent late-LTP (Tsokas et al., 2005; Tsokas et al., 2016, Hsieh et al., 2017). For rat hippocampal slices deliver the tetanus at 70% of the spike threshold (Tsokas et al., 2005; Hsieh et al., 2017), and for mouse slices use 25% of the spike threshold (Tsokas et al., 2016). The maximum slope of the rise of the fEPSP can be analyzed on a PC using the WinLTP data acquisition program (Anderson and Collingridge, 2007). 

    Notes

    1. Comparison of interface and submersion chamber: oxygenation and efficacy of aPKC inhibitor ZIP on synaptic transmission
      Most studies examining long-term synaptic plasticity in hippocampal slices, employ Oslo-type interface recording chambers, rather than submerged chambers, because the former are believed to provide superior oxygenation and preservation of normal synaptic function (Khurana and Li, 2013). We tested this assumption, by using a galvanic dissolved-oxygen probe (Atlas Scientific KIT-106; zinc anode, silver cathode, 15% sodium tetraborate/15% sodium chloride electrolyte; polyethylene membrane) to measure the dissolved oxygen (DO) concentration in the Oslo-type interface recording chamber and in the same chamber used in submersion mode with flow rates ranging from 2 to 15 ml/min.
        The dissolved oxygen probe was calibrated using distilled water and ambient air, as described by the manufacturer. The accuracy of the instrument was further tested by measuring dissolved atmospheric oxygen in ACSF (14,000 μS/cm) at room temperature (23.5 °C). The average value (8.1 ± 0.01 mg/L; n = 5) was in agreement with previously reported data for atmospheric dissolved oxygen equilibrium at different temperatures and salinity conditions (Radtke et al., 1998).
        Air from a medical air cylinder, or 100% oxygen (both from TW Smith) was then bubbled through the gas dispersion ring (or the ceramic aerators) of the water jacket of the Oslo-type chamber at 31.5 °C, and the concentration of dissolved oxygen was measured inside the recording chamber, under interface conditions, with the probe clamped on a micromanipulator and held a few millimeters above the surface of the mesh (where the meniscus surrounding the slice is normally formed). A standard curve of oxygen saturation vs. concentration was thus generated for the tip of the probe moistened with ACSF (6.9 ± 0.3 mg/L at 21% O2; 31.1 ± 0.8 mg/L at 100% O2; n = 5; linear fit through 0, r2 = 0.99). With 95% O2-5% CO2 bubbling through the water jacket, the oxygen concentration immediately above the slice at the interface was 29.6 ± 0.7 mg/L (95.1 ± 2.3% saturation; n = 5).
        The Oslo-type chamber was then converted into a submersion chamber: the suction was raised to increase the level of the superfusate inside the recording bath, the oxygenation via the aerators in the water jacket was turned off, and the flow rate was increased to obtain oxygen concentration/saturation measurements at 2, 5, 10 and 15 ml/min. Under submersion conditions, and immediately above the position where the slice would be in an actual experiment, the following oxygen concentration (mg/L) and % saturation measurements were obtained (n’s = 4)—2 ml/min: 19.6 ± 0.9 (62.5 ± 2.8%); 5 ml/min: 22.7 ± 1.4 (72.4 ± 4.6%); 10 ml/min: 25.5 ± 1.7 (81.6 ± 5.7%); and 15 ml/min: 26.9 ± 1.9 (86.3 ± 6.2%). These measurements are in agreement with values reported for different types of submersion chambers described elsewhere, using carbon-fiber microelectrodes, DO optical sensors, polarographic cells (i.e., amperometric Clark electrodes), or a colorimetric chemical (indigo carmine) methods (Hájos et al., 2009; Dondzillo et al., 2015; Weng et al., 2018).
        These results demonstrate that the oxygen saturation in the superfusate of submersion chambers (see, for example, Volk et al., 2013) is substantially lower than in the humidified atmosphere that envelops slices in Oslo-type interface chambers, especially at the flow rates commonly used (72.4 ± 4.6% for a submersion chamber with flow rate 5 ml/min, compared to 95.1 ± 2.3% for an Oslo-type interface chamber). The effects of lower oxygen saturation on slice health might be further exacerbated when the slices are recovered under submerged conditions after preparation (as in Volk et al., 2013).
        We next asked whether the differences of oxygenation obtained in interface vs. submerged chambers have physiological effects on synaptic physiology. We specifically addressed whether chamber design might explain a difference that has been reported in the effects of the atypical PKC inhibitor ZIP on basal synaptic transmission. Whereas consistently ZIP reverses late-LTP in all published studies in which it has been studied (see, for example Ling et al., 2002; Sajikumar et al., 2005; Pastalkova et al., 2006), in most of these studies the effect of the PKMζ inhibitor is specific to potentiated synaptic pathways with no effect on basal, untetanized synaptic pathways. This specificity of the inhibitor to potentiated and not basal synaptic transmission is identical to results obtained with applications of the agent to LTP and basal synaptic transmission recorded in vivo (Pastalkova et al., 2006; Madroñal et al., 2010). However, one study reported that ZIP had effects on both late-LTP and baseline synaptic transmission (Volk et al., 2013). This study, however, was unusual in that the late-LTP recordings and basal synaptic transmission were recorded in hippocampal slices held in a submerged chamber.
        We therefore directly compared the effects of ZIP on LTP and baseline synaptic transmission in hippocampal slices recorded in an interface and in a submersion chamber. To keep all other parameters as constant as possible, the interface chamber slices recovered in the same chamber in which their recordings take place; whereas the submersion chamber slices were placed in an interface chamber, which was then immediately converted into a submersion chamber by increasing the flow rate and bath level of the ACSF solution, and the slices were then allowed to recover for another 90 min (the total recovery time of both sets of slices in the two experiments is kept equal).
        As seen in Figure 21, in slices recovered and recorded in an interface chamber, ZIP specifically reverses late-LTP maintenance in the tetanized pathway without affecting basal neurotransmission in the untetanized pathway. In contrast, in slices recorded under submerged conditions, ZIP reverses LTP maintenance, eventually below baseline, and also has an effect on an independent untetanized pathway. This effect of ZIP on the untetanized/basal pathway was similar to that seen in Volk et al. (2013).
        A plausible explanation for this difference is that there is a pool of atypical PKC involved in neuroprotection from hypoxia (see Tian et al., 2008) that is separate from the pool of PKMζ that maintains late-LTP. Because the oxygenation conditions in an interface chamber are superior to those for submerged slices, the effect of ZIP on basal neurotransmission in a submersion chamber may reflect the effects of the inhibitor on the neuroprotective role of atypical PKC that is induced by the relatively hypoxic conditions of the slice (Tian et al., 2008). Alternatively, the low-oxygen conditions of submerged chambers may have induced hypoxic LTP in the untetanized synapses, rendering them sensitive to ZIP. Importantly, as mentioned above, the specificity of ZIP to potentiated and not basal synaptic transmission is seen in vivo in the hippocampal perforant path dentate gyrus input (Pastalkova et al., 2006), hippocampal CA3-CA1 (Madroñal et al., 2010), and layer 4 primary visual cortex (Cooke and Bear, 2010). Thus, high-oxygen interface chambers, but not low-oxygen submerged chambers appear to produce conditions of synaptic transmission and plasticity that more closely reflect in vivo conditions.
        In addition to inferior oxygenation conditions, another factor that may have contributed to the artifactual response to ZIP of the untetanized pathway in Volk et al. (2013) is the intensity of the conditioning stimulus used to induce late-LTP. The intensity of the Volk et al. (2013) protocol, which was delivered at 75% of the maximum EPSP response, is appropriate for rat Schaffer collateral/commissural-CA1 synaptic stimulation, but is much higher than what is normally used to induce LTP in mouse hippocampal slices, which is typically between 25% of the spike threshold, as in Tsokas et al. (2016), and 50% of maximum EPSP in Abel et al. (1997), Nguyen et al. (2000), Barco et al. (2002), Opazo et al. (2003), Selcher et al. (2003), Kelleher et al. (2004), Costa-Mattioli et al. (2005), Antion et al. (2008), Pavlowsky and Alarcon (2012), Gruart et al. (2012), Li et al. (2017), Puzzo et al. (2017), Amorim et al. (2018). Such strong repetitive synaptic stimulation in Volk et al. (2013) may have further undermined neuronal health, thereby contributing to the artifactual drop below the baseline of the EPSP in the untetanized pathway in response to ZIP.


      Figure 21. Brain slice late-LTP experiments in an interface vs. submersion chamber. A. ZIP (5 μM) applied during the maintenance phase of late-LTP reverses potentiation without effect on the untetanized pathway (from Tsokas et al., 2016; n = 5). B. In contrast, in slices (n = 3) that have recovered in an interface chamber (for equal time as the slices in A), which was then converted into a submersion chamber by increasing the bath level and flow rate of the ACSF, 5 μM ZIP not only reverses LTP in the tetanized pathway but also decreases basal synaptic transmission in the untetanized pathway. This difference may be due to the superior oxygenation conditions in interface chambers (oxygen saturation is 95.1 ± 2.3% in an Oslo-type interface chamber, compared to 72.4 ± 4.6% in a submersion chamber with a typical flow rate of 5 ml/min).

    2. Protection from excitotoxicity
      In order to protect from excitotoxicity during the slice-making process we recommend using ice-cold high magnesium/low calcium ACSF solutions and performing the slice preparation procedure inside a 4 °C cold room.
        Lowering the temperature decreases the metabolic rate of cells thus reducing their energy consumption, allowing the cells to survive ischemia during slice preparation. In addition, we recommend using a special, non-physiological ACSF during dissection (Dissection ACSF) that contains 10 mM magnesium and 0.5 mM calcium. Both these departures from physiological CSF are known to reduce excitotoxicity (Feig and Lipton, 1990; Sacktor et al., 1993; Wang and Kass, 1997; Ting et al., 2018).
    3. mp6 micropump noise
      There are two sources of noise in the system: (a) noise from the electrical signal of the OEM, and (b) noise generated in the solution because of the vibration of the ionic ACSF by the pump actuators. These sources of noise can be completely silenced by (a) making a metal enclosure for the circuit and mini-Faraday cages for the pump “sandwich” arrays, and (b) grounding the recirculating ACSF both at the inflow to the bath and at the suction, using T-shape tubing connectors with a silver chloride wire connected to the main ground of the rig.
    4. Passive check valves mp-cv
      To eliminate potential back flow of the ACSF owing to differential pressure between the pump inlet and the outlet (due to differences, for example, between the height of the recording chamber and the height of the ACSF reservoir, or the connecting tubes), use a passive check valve encased in stainless steel manufactured by Bartels Mikrotechnik (mp-cv). The valve, which will influence the volume flow of the micropump, should be placed between the micropump and the outflow reservoir. Figure 15C shows four such mp-cv check valves connected to the array of four mp6 micropumps controlling the suction of the interface recording chamber.
    5. Metal enclosure for the piezoelectric pump system
      For safety reasons (to protect against electric shock from the OEMs), as well as to eliminate the noise produced by the OEMs and to protect the circuit from damage, the completed circuit of Figure 14 should be placed inside a metal enclosure that is grounded. The enclosure should have an opening for the ribbon cable of the Breakout Board connecting the micropump assemblies, as well as openings for the power supply cables, the two USB cables of the Arduinos, the master switch of the circuit, and the BNC cable connecting the top Arduino to the ADC board.
    6. Hybrid persistaltic/piezoelectric pump arrangement
      A simplified alternative to the recirculation device described here is to use piezoelectric pumps only for the aspirating the recirculation ACSF, and a peristaltic pump to provide the inflow to the chamber. In this arrangement, a peristaltic pump with a two-channel pump head would control the inflow (alternatively, a four-channel pump head can be used, if the interface chamber supports two independent baths for slices). One piezoelectric array (or two, depending on the number of baths supported by the interface chamber) would control the aspirator, with each array consisting of four mp6 micropumps driven by the suction part of the circuit (i.e., the top four OEMs) at the maximum flow rate.
    7. Pre-flushing the dead volume of the perfusion system before switching solutions
      When 95% O2-5% CO2-aerated ACSF stays in polyethylene or Tygon tubing for a long time before being perfused onto slices, it degases and becomes hypoxic. The loss of carbon dioxide also alters the pH of the standard bicarbonate buffer of the ACSF. It is therefore important to “pre-flush” the dead volume in the perfusion system before switching solutions.
        In the experiments described in Tsokas et al. (2016), two manual three-way valves in series allowed pre-flushing the dead volume in the tubing of the inflow mp6 micropump assemblies by switching the upstream three-way valve from the idle peristaltic pump (OFF) to the idle mp6 micropump (ON), and switching the downstream three-way valve so that the outflow from the micropump flows directly into waste (ON), rather than into the slice chamber (OFF). In this configuration, turning the micropump on for 10 seconds before switching it back off allows the dead volume to clear. Following this procedure, first turn the downstream valve’s outlet to the waste to OFF, before turning the mp6 micropump back on to resume operation of the perfusion system.
        WinLTP 2.30 has four perfusion line controls (Slow0, Slow1, Fast0, and Fast1) that can be used for controlling piezo steppers and pinch valves for automatic changing of bath perfusion. For an excellent discussion of how to use these features with commercially available pinch valve systems for automated pre-flush perfusion of slices, see Chapter 10 of the WinLTP 2.30 manual (Anderson, 2018).
    8. The mp6 micropumps are sensitive and will be severely damaged if liquid is forced into them by applying pressure with a syringe. If the micropumps fail to draw liquid when initially turned on (usually due to incomplete purging of their contents during previous use), it is best to connect the inlet of the mp6 to proper tubing attached to the tip of a vertically positioned 20 ml syringe barrel (i.e., without the plunger, and with its tip pointed towards the ground). Turn the micropump on, pour a few mL of water into the barrel of the syringe, and allow gravity to assist the micropump’s vibrating piezoelectric diaphragms in clearing the chambers of the pump.

    Recipes

    1. 10x Stock Dissection ACSF Buffer (maintain at 4 °C) (Table 1)

      Table 1. Recipe for preparing 10x Stock Dissection ACSF Buffer (maintain at 4 °C)
      Reagent
      Stock Concentration (10x)
      For 1 L
      NaCl
      1.25 M
      73.20 g
      KCl
      25 mM
      1.86 g
      NaH2PO4
      12.5 mM
      1.50 g
      NaHCO3
      260 mM
      21.84 g

    2. 10x Stock Recording ACSF Buffer (maintain at 4 °C) (Table 2)

      Table 2. Recipe for preparing 10x Stock Recording ACSF Buffer (maintain at 4 °C)
      Reagent
      Stock Concentration (10x)
      For 1 L
      NaCl
      1.18 M
      69.00 g
      KCl
      35 mM
      2.61 g
      NaH2PO4
      12.5 mM
      1.50 g
      NaHCO3
      240 mM
      20.15 g

    3. 1x Dissection ACSF Buffer (Table 3)

      Table 3. Recipe for preparing 1x Dissection ACSF Buffer (prepare on the day of the experiment)
      Reagent
      Working Concentration (1x)
      For 1 L
      CaCl2
      0.5 mM
      0.5 ml of 1 M CaCl2 solution
      MgCl2
      10 mM
      10 ml of 1 M MgCl2 solution
      D-(+)-Glucose
      11 mM
      1.98 g
      10x Dissection ACSF

      100 ml
      Water

      889.5 ml

    4. 1x Recording ACSF Buffer (Table 4)

      Table 4. Recipe for preparing 1x Recording ACSF Buffer (prepare on the day of the experiment)
      Reagent
      Working Concentration (1x)
      For 1 L
      CaCl2
      2.5 mM
      2.5 ml of 1 M CaCl2 solution
      MgCl2
      1.3 mM
      1.3 ml of 1 M MgSO4 solution
      D-(+)-Glucose
      15 mM
      2.71 g
      10x Recording ACSF

      100 ml
      Water

      896.2 ml

    Acknowledgments

    This piezoelectric micropump recirculation system and ODN delivery protocol was originally used in Tsokas et al. (2016). This work was supported by NIH grants R21NS091830 and R01MH099128 (AAF) and R37MH057068, R01MH115304, R01MH53576, R01DA034970, and the Lightfighter Trust (TCS). We thank Randy Andronica for technical assistance. The authors would like to thank Davide B. R. Bianchi (Boston University, Boston, MA) and Zoe B. Ravera (Ethical Culture Fieldston School, New York, NY) for building prototypes based on this protocol in order to test its accuracy. Panayiotis Tsokas is an Alexander S. Onassis Public Benefit Foundation Scholar.

    Competing interests

    The authors declare that no competing interests exist.

    Ethics

    Animal experimentation: The experiment in Tsokas et al. (2016), were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#11-10274, #12-10298, #13-10363 of SUNY Downstate Medical Center or #15-1459 of New York University). The protocols were approved by the Institutional Animal Care and Use Committee of SUNY Downstate Medical Center (Animal Welfare Assurance Number: A3260-01) and New York University (Animal Welfare Assurance Number: A3317-01). All surgery was performed under either ketamine and dexmedetomidine, isoflurane, or sodium pentobarbital anesthesia, and every effort was made to minimize suffering.

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简介

长期突触可塑性的分子机制的阐明受到两个因素的阻碍:一是核心可塑性分子长期丢失后可能发生的代偿作用,二是体内不可能再生可塑性的条件。在这里我们描述了一种新的快速抑制基因表达的方法,通过反义寡核苷酸(odns)应用于啮齿动物脑切片的“oslo型”接口室。该方法有三个优点:1)快速阻断靶蛋白的新合成,避免遗传补偿;2)脑片的有效氧合,这对于在体内复制长期突触可塑性条件至关重要,3)循环系统,只使用少量的溶液(<5毫升),减少长期实验所需的试剂量,持续数小时。该方法采用压电微泵的定制再循环系统,首次用于Tsokas等人于2016年在长期增强(LTP)过程中对蛋白激酶Mζ(PKMζ)合成的急性翻译阻断。在该研究中,应用反义odn可迅速阻止pkmζ的合成,并阻断晚期ltp,而不会诱导pkcζ/pkmζ基因敲除小鼠中其他蛋白激酶c(pkc)亚型的补偿。此外,我们还发现,在低氧浸没型腔中,非典型PKC抑制剂、Zeta抑制肽(ZIP)的应用可导致基线突触传递不稳定,但在高氧、奥斯陆型界面电生理室中,药物逆转晚期LTP而不影响基线突触传递。这种比较表明,界面室,但不是淹没室,再现Zip 体内>的影响。因此,该协议结合了新的特异性蛋白质合成的能力,用于研究长期突触可塑性,同时保持了突触传递的特性,再现了体内>与长期记忆相关的条件。
【背景】活体外急性脑切片是一种在分子、细胞和电路水平上研究神经功能的有效实验模型。基因敲除技术的进步也极大地促进了转基因小鼠的产生。这些突变小鼠的海马切片已经成为研究人类精神和神经疾病动物模型中正常突触可塑性及其功能障碍最广泛使用的实验范式之一。



然而,一些研究表明,基因敲除引起的基因补偿是一种普遍现象(回顾见El Brolosy and Stainier,2017)。基因敲除后相关基因的上调可能减弱或完全补偿缺失基因的功能,导致实验结果可能被曲解。



通过简单应用反义寡核苷酸(odns),可以通过急性阻断靶基因产物的从头合成来避免这种补偿。ODN是短的单链合成DNA分子,通常用硫代磷酸酯键修饰以增强稳定性。odns可以与特定mrna的互补区域结合,通常位于翻译起始点附近(图1a,顶部,aug),并在物理上阻断核糖体沿着mrna移动的能力,阻止蛋白质的合成。由于odns的作用是迅速的,随着基因缺失时间的延长,改变其他基因调控的可能性降低。可能会出现潜在的非靶向效应,但可以通过适当的ODN设计和采用ODN和缺乏靶向mRNA的敲除物的药物遗传学实验最小化(Tsokas等人,2016年)。



以往的研究大多采用odn来抑制脑切片中的基因表达,然而,首先通过颅内或脑室注射将odn注入大脑,然后在注射后不久制备组织切片(matthies等,1995;guzowski等,2000;Garcia Osta等人,2006年)。尽管注射可以有效地将odn导入大脑,但它本身往往会损伤神经组织,导致注射部位附近脑切片的记录不理想。此外,注射以高浓度的丸状物的形式传递化合物,然后必须在切片制备之前通过完整动物的脑组织扩散。结果,实验者无法精确控制记录时切片中化合物的浓度。如果需要在丸注射剂中高浓度的药物,这反过来又会导致药理学特异性的问题。



本方案中所述的方法使得用反义odn灌注脑片与应用任何其他可溶性试剂或药物一样容易。因此,该技术可能优于颅内或脑室内注射然后制备脑切片的标准方法。此外,电生理记录是在一个“奥斯陆式”接口脑切片记录室。对于脑切片的长期记录,接口室比潜水室更可取,因为它们提供了更好的氧合(详细讨论见注1)。该方案首次在Tsokas等人(2016年)中使用,在不诱导其他非典型蛋白激酶C(PKC)的遗传补偿的情况下,特异性地阻断蛋白激酶Mζ(PKMζ)的新合成,以应对强直性传入刺激(高频刺激,HFS)。同型,pkc/λ,出现在pkmζ-无小鼠中。事实上,在这些突变小鼠中,由于pkc/λ在很大程度上补偿了pkmζ的正常生理功能,长期增强(ltp)和记忆形成在很大程度上是完整的(tsokas等人,2016年)

不可培养细菌是指那些在原生境中繁殖,但在正常的实验室培养基和条件下不能生长或繁殖的细菌。分子技术揭示了这些未培养细菌在功能多样性和产生次生代谢产物的潜力方面的意义。为了实现这些益处,科学家们尝试使用TnWistern平板、光镊、激光显微切割、微生物反应器和扩散生物反应器在实验室中分离和培养不可培养的细菌。然而,这些技术仍然不足以解决培养不可培养细菌的困难。因此,在实验室条件下开发新的培养方法是非常必要的。扩散生物反应器是一种膜结合室,允许微生物通过在天然环境中提供过量的营养物质和信号化合物而在其原生环境中增殖。本文提出了一种高效可靠的扩散生物反应器的构建方法,并将其应用于实验室中不可培养土壤细菌的分离培养。

关键字:长时程增强, LTP, 压电泵, PKMζ, PKM-ζ, ZIP

材料和试剂

  1. 双面胶带(亚马逊,型号:QK-8285b0134cufu0_-loc)
  2. 单刃刀片(Gem Personna,电子显微镜科学,目录号:71972)
  3. 双刃刀片(电子显微镜科学,目录号:71998)
  4. Pyrex量筒2000毫升(康宁,大众汽车,目录号:3022-2L)
  5. Pyrex量筒1000毫升(康宁,大众汽车,目录号:3022-1L)
  6. Pyrex刻度量筒100毫升(康宁,大众汽车,目录号:70075-100)
  7. Pyrex培养皿90mm(康宁,VWR,目录号:7740)
  8. Pyrex介质储存瓶,1L(康宁,大众汽车,目录号:1395-1L)
  9. 派热克斯气体分散管(康宁,大众汽车,目录号:39533-12C)
  10. 派瑞克斯刻度烧杯,250毫升(康宁,VWR,目录号:1000-250)
  11. Pyrex刻度烧杯,50 ml(康宁,VWR,目录号:1000-50)
  12. Pyrex刻度烧杯,20 ml(康宁,VWR,目录号:1000-20)
  13. Vwr锥形瓶,250毫升锥形瓶(Vwr,目录号:75804-646)
  14. 15ml锥形管(VWR62406-200)
  15. 手术刀手柄3(精细科学工具,目录号:10003-12)
  16. 手术刀刀片10(精细科学工具,目录号:10010-00)
  17. 外科剪刀硬剪(精细科学工具,目录号:14054-13)
  18. 超细波恩剪刀(精细科学工具,目录号:14084-08)
  19. Friedman Pearson-Rongers(精细科学工具,目录号:16221-14)
  20. 抹刀(VWR,目录号:82027-490)
  21. 椭圆匙抹刀(VWR100493-952)
  22. 骆驼毛刷1(Ted Pella,Inc.,目录号:11859)
  23. 昆虫针(精细科学工具,目录号:26000-50)
  24. 沃特曼5级滤纸55毫米(沃特曼,VWR,目录号:1005 055)
  25. Whatman 5级滤纸90毫米(Whatman,Vwr,目录号:1005 090)
  26. 移液管(Fisherbrand,Fisher Scientific,目录号:13-711-7M)
  27. 内径为1.3 mm的油管接长件(Cole Parmer,目录号:SK-95809-32)
  28. Tygon管,内径1.6 mm(Cole Parmer,目录号:SK-06407-33)
  29. 聚四氟乙烯/聚四氟乙烯管,内径1.6 mm(Cole Parmer,目录号:SK-95231-00)
  30. 三通阀(Cole Parmer,目录号:SK-30600-02)
  31. 内外镀金销(精细科学工具,目录号:19003-01和19-003-00)
  32. 氯化钙溶液,1 m(CaCl2)(Teknova,目录号:C0477,室温)
  33. 氯化镁溶液,1 m(mgcl2)(Teknova,目录号:M0304,室温)
  34. 1 m硫酸镁溶液(mgso4)(Teknova,目录号:M3003,室温)
  35. 氯化钾(KCl)(Fisher Scientific,目录号:P9541-1kg,室温)
  36. 碳酸氢钠(NaHCO3)(Sigma-Aldrich,目录号:S5761-500g,室温)
  37. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S271-3,室温)
  38. 一元磷酸钠(NaH2Po4)(Sigma Aldrich,目录号:S5011-500g,室温)
  39. D-(+)-葡萄糖(Sigma-Aldrich,目录号:G7528-1kg,室温)
  40. 异氟烷溶液(C102CLF5O)(Henry Schein动物健康,目录号:029404,室温)
  41. 根据Tsokas等人(2016年),Hsieh等人(2017年)(基因链接,纽约州霍桑市,4°C),每端三个末端碱基上带有5'-3'磷酸键的反义加扰ODN(纯化的反相药筒),以防止核酸酶降解。
  42. 100%氧气(TW Smith)
  43. 95%氧气-5%二氧化碳(TW Smith)
  44. 10x库存解剖ACSF缓冲液(见配方)
  45. 10x库存记录ACSF缓冲区(见配方)
  46. 1x解剖ACSF缓冲液(见配方)
  47. 1x记录ACSF缓冲区(见配方)

设备

  1. 4°C冷藏室
  2. -70°C冰箱:Thermo Scientific Forma 88000系列-86°C(Thermo Scientific,型号:88600D)
  3. 水浴:Fisherbrand ISOtemp数字控制水浴202型(Fisher Scientific,型号:15-462-2Q)
  4. 康宁带盖圆形冰桶,4L(Fisher Scientific,目录号:07-210-123)
  5. “奥斯陆式”接口脑片记录室(精细科学工具,目录号:21000-02)
  6. 华纳TC-344C双通道温度控制器(华纳仪器,目录号:64-2401)
  7. 带臂架的解剖双目立体变焦显微镜(尼康,莫雷尔仪器公司,目录号:SMZ800N)
  8. 光纤光源(Dolan Jenner Miledusb1 MI-LED-US-B1)
  9. 双鹅颈光纤组件(Dolan Jenner EEG3736 002144113036)
  10. 1600型神经探针放大器(A-M系统,目录号:680100)
  11. 1600型前台(A-M系统,目录号:681500)
  12. ADC连接器盒(国家仪器,目录号:BNC-2090A)
  13. ADC PCI总线卡(国家仪器,目录号:PCIE-6323)
  14. 戴尔Optiplex 3040(亚马逊,目录号:b01e8qrsag)
  15. iso-flex刺激器(ampi,目录号:iso-flex)
  16. Narishige MX-2微操作器
  17. Narishige MM-3微型操作器
  18. 火焰/棕色吸管拉拔器(萨特仪器,目录号:P-97)
  19. McIlwain组织切碎器(McIlwain实验室,Ted Pella,Inc.,目录号:10180)
  20. 塑料盘(Ted Pella,Inc.,目录号:10180-01)
  21. 啮齿动物断头台(WPI,世界精密仪器,目录号:DCAP)
  22. 异氟醚汽化器(vss,目录号:300系列)
  23. 麻醉诱导室(徕卡生物系统,目录号:39481051/3)
  24. 六个MP6 OEM控制器(MP6 OEM,Bartels Mikrotechnik GmbH,Servoflo目录号:MP6 OEM)
  25. 六台MP6微型泵(MP6,Bartels Mikrotechnik GmbH,Servoflo目录号:MP6)
  26. 六个被动止回阀MP-Cv(Bartels Mikrotechnik,Servoflo,目录号:MP-Cv)
  27. 六个FCC连接器(Molex;Digi键,目录号:0039532045)
  28. 两个Arduino UNO微控制器(Adafruit Industries,目录号:782-a00066)
  29. 两个8通道双向逻辑电平转换器(Adafruit Industries,目录号:TXB1008)
  30. A&B型覆盆子皮用装配式皮皮补板+26针带状电缆(Adafruit,目录号:914)
  31. Arduino屏蔽堆叠头(R3兼容)(Adafruit,目录号:85)
  32. 公/公跳线(Adafruit,目录号:7581956)
  33. 跨接导线套件-预切割和直角弯曲,700根(Sparkfun电子产品,目录号:PRT-14671)
  34. MC001阿尔法导线多导体电缆(MOUSER,目录号:602-1102-100)
  35. WBU-206踏板31 mm x 175 mm x 230 mm(纽瓦克电子,目录号:18M7115);备选方案:数字340-002-1(数字键,目录号:1286-1220-ND)
  36. 10k欧姆15转金属陶瓷电位计/微调器(Radioshack,产品目录号:2710343)
  37. RS Pro 1440976,交流-直流桌面电源,5伏直流,7.5安(Allied Electronics,目录号:71259700)
  38. 单刀双掷开关(RadioShack,目录号:2750011)
  39. 六个倒钩Y形管接头(Servoflo,目录号:MP-Y)
  40. 热缩管(Mouser,目录号:650-Ver332sp)
  41. WELLER WTCPT焊接站(数字键,目录号:WTCPT-ND)
  42. 焊料(数字键,目录号:KE1106-ND)
  43. 加热枪:AlphaWire Fit Gun-1(数字键,目录号:FGUN1NC032-ND)
  44. 万用表fluke 177(数字键,目录号:614-1020-nd)
  45. USB电缆(亚马逊,ASIN:b003zzh2u8)
  46. 九个定制有机玻璃垫片(MSC工业供应,目录号:63388953)
  47. 六个定制软木垫垫片(来自亚马逊的软木垫片,ASin:b001acmbo0)
  48. 平头2-56¾英寸螺钉:(MSC工业供应,目录号:40914715)
  49. 铜33规格,0.011英寸线径,16 x 16目(MSC工业供应,目录号:52427291)
  50. 尼龙搭扣1“宽背胶钩环卷(MSC工业供应,目录号:67127480)
  51. 夹具(Fisher Scientific,05-769-7Q;S13743;02-215-466;02-300-206)
  52. 可选:溶解氧试剂盒(Atlas Scientific,Atlas Scientific,目录号:Kit-106)

软件

  1. WinLTP(WinLTP有限公司和布里斯托尔大学,https://www.winltp.com/“target=”\u blank“>https://www.winltp.com)
  2. Arduino软件(IDE)(Arduino,https://www.arduino.cc/en/main/software“target=”\u blank“>https://www.arduino.cc/en/main/software)
  3. Atlas Scientific溶解氧原始设备制造商样品代码(https://www.atlas-scientific.com//u files/code/oem-do sample code.pdf)

程序

第一部分:ACSF再循环系统总成
< BR> < /强>

  1. 微型泵控制电路的组装
    有关带有彩色编码电缆的简化示意图,请参阅图2A。有关电路组装的详细逐步照片,请参阅图4-14。有关列出MP6微型泵控制电路所需所有连接的表格,请参见图3。
    < BR>
    图3。列出MP6微型泵控制电路所需的所有连接的表 < BR>
    图4。组装MP6微泵控制电路的步骤1-4。有关详细信息,请参阅文本。
    < BR>
    1. 为了安装所有必要的部件,需要一个带有3个端子排和63行10个连接点的接线板(图4)。这些面包板包括数字340-002-1或Wisher WBU-206。每一个都有5条配电母线和3条接线板。拆下(拧下)最右侧的配电总线和相邻的端子排。
    2. 用螺钉或尼龙搭扣将两个Arduinos连接起来-理想情况下是交错连接,以便轻松访问两个USB端口,如图4所示。
    3. 将5伏直流电源连接到垫板的水平分布总线上(图4,用红色“加号”和黑色“减号”表示)。包括一个简单的切换spst按钮“主开关”(未显示),用于将电源切换到电路。
    4. 将所有垂直配电母线的电源和接地分别连接至水平配电母线的5 V和接地(图4)
      < BR>
      图5。组装MP6微泵控制电路的步骤5-6。有关详细信息,请参阅文本。
      < BR>
    5. 在中间(现在是最右边)的接线板上连接两个10K欧姆电位计,并将它们连接到电源和接地,如图5所示。< BR> 注意:旋转雨刮器和改变每个电位计中电阻的螺钉应面朝对方,从而为将用于在步骤6中进行调整的微型螺丝刀留出足够的间隙。>
    6. 使用小螺丝刀,调整电压,使顶部电位计的刮水器电压为1.3 V,底部为1.8 V。
      注意:为了安全起见,在继续执行协议的下一步时,应断开电源。>
      < BR>
      图6。组装MP6微泵控制电路的步骤7-8。有关详细信息,请参阅文本。
      < BR>
    7. 在包括两个电位计的接线板上,还安装两个8通道逻辑电平转换器(图6)。将每个逻辑电平转换器上的VCCB连接到5 V,接地连接到接地(图6;有关引出线图和电压信息,另请参阅图2A)。
    8. 将顶部逻辑电平转换器上的VCCA和OE连接至1.3 V,底部转换器上的连接至1.8 V(参见图6中的灰色跨接导线;有关电压和引脚信息,另请参见图2A)。
    9. 连接26针组装的圆周率鹅卵石突破板。使用屏蔽堆叠头来提升转接板,从而为第三和第四个MP6 OEM控制器留出足够的间隙,这些控制器将在下一步(步骤10)中连接,并直接与转接板相邻(见图15d)。
      < BR>
      图7。组装MP6微型泵控制电路的步骤9-14。有关详细信息,请参阅文本。
      < BR>
    10. 如图7所示,将六个MP6 OEM控制器连接到试验板最左边的端子板上(另请参阅图2A)。
      注:底部的两个MP6 OEM控制器将提供接口室槽的流入管线(对网格的顶部和底部进行灌注),其流量可由底部的Arduino Uno调节,Arduino Uno连接到引脚2(步骤22中添加的青色)(图2,以及图15a和15b)。前四个原始设备制造商连接到提供抽吸的四泵阵列(图15c),它们的流量是恒定的(设置为最大值)。对于这些原始设备制造商,针脚2和针脚3短路,并且没有收到来自底部Arduino UNO的任何输入(图2A)。还要注意,在所有六个原始设备制造商中,针脚5和针脚10都未使用。>
    11. 所有6个MP6原始设备制造商上的短插脚11和12,使用由切断的跨接导线制成的U形环,长度足以连接相邻垫板排上的两个触点(参见图7中的六个尖星号;有关插脚的编号,请参见图2A)。
    12. 同样,四大原始设备制造商中的短插脚2和3(参见图7中的五个尖星号;插脚编号参见图2a)。
    13. 将接地(黑色)连接到插脚1,标记有所有6个MP6原始设备制造商的白点(图7;有关插脚编号,请参阅图2A)。
    14. 将5 V(红色)连接到所有6个MP6原始设备制造商的插脚14,如图7所示(有关插脚的编号,请参阅图2A)。
      < BR>
      图8。组装MP6微型泵控制电路的步骤15。有关详细信息,请参阅文本。
      < BR>
    15. 将顶部逻辑电平转换器A1、A2、A3、A4、A5、A6的1.3 V(黄色)连接到所有6个MP6原始设备制造商的引脚4(图8;图2A的示意图中仅显示了两个这样的连接)。
      < BR>
      图9。步骤16组装MP6微泵控制电路。有关详细信息,请参阅文本。
      < BR>
    16. 将底部逻辑电平转换器A1、A2、A3、A4、A5、A6的1.8 V(橙色)连接到所有6个MP6原始设备制造商的引脚13。(图9;图2a的示意图中仅显示了两个这样的连接)。
      < BR>
      图10。组装MP6微泵控制电路的步骤17-18。有关详细信息,请参阅文本。
      < BR>
    17. 将插脚6(棕色)连接到已装配的圆周率圆片叉接线束左侧的插脚1、3、5、7、9、11(图10,第12页)。
    18. 将插脚7(绿色)连接到已装配的圆周率圆片叉接线束(图10)的插脚2、4、6、8、10、12上。
      < BR>
      图11。组装MP6微泵控制电路的步骤19-20。有关详细信息,请参阅文本。
      < BR>
    19. 将插脚8(蓝色)连接到已装配的圆周率圆片叉接线束(图11)的右侧插脚2、4、6、8、10、12上。
    20. 将插脚9(紫色)连接到已装配的圆周率圆边叉接线束(图11)左侧的插脚1、3、5、7、9、11上。
      < BR>
      图12。步骤21组装MP6微泵控制电路。有关详细信息,请参阅文本。
      < BR>
    21. 将顶层逻辑电平转换器的输入端B1、B2、B3、B4、B5、B6分别连接到顶层Arduino UNO的数字管脚2、3、4、5、6、7,如图12所示(白色电缆)。
      < BR>
      图13。步骤22组装MP6微泵控制电路。有关详细信息,请参阅文本。
      < BR>
    22. 将底部逻辑电平转换器的输入端B1、B2、B3、B4、B5、B6分别连接到顶部Arduino UNO的数字管脚8、9、10、11、12、13,如图13所示(洋红色电缆)。
      < BR>
      图14。步骤23并完成MP6微型泵控制电路总成的概述。有关详细信息,请参阅文本。
      < BR>
    23. 将底部Arduino UNO的输出9和10(PWM0和PWM1)连接到底部两个OEM的引脚2(青色)(图14)。
    24. 将ADC的一个数字输出连接到顶部Arduino UNO的TX 1和GDN(图2A)。此连接将按照winltp程序的指示打开和关闭由mp6泵控制的再循环灌注。
    25. 使用USB连接将两个Arduino UNO单元连接到计算机。
    26. 将底部Arduino的GDN连接到电生理设备的中央接地。(图2a.顶部Arduino通过ADC接地,参见步骤24)。
    27. 将如图18所示的程序复制并加载到底部Arduino控制四个流出微泵(微控制器2)。
    28. 将图19所示的程序复制并加载到Arduino控制两个流入微泵(微控制器1)中。
    < BR>
  2. MP6微泵阵列的组装(见参考文献29)
  1. 如图2b和15e所示,组装将六个Molex连接器(每个MP6微型泵一个)连接到26针带状电缆的六根电缆。图2b和2c的颜色方案作为指南。使用四个镀金公针和一个32AWG四线多导体电缆(例如MC001阿尔法线多导体电缆)将Molex连接器的四个针连接到带状电缆。每个Molex连接器和相关的多色电缆将插入带状电缆的四个插座中(电线排列,从左上角插座顺时针方向:棕色、紫色、蓝色、绿色;对所有六个Molex连接器重复此操作)。在带状电缆的26个插座中,仅使用24个(6 x 4)。
    注:忽略图15a、15b、15c、15e和15f中α线多色电缆的颜色,这些颜色与图2b的颜色方案无关。>
  2. 如图2c所示放置两个部件,MP6/MP6 PP面朝上(从上面可以看到其公司徽标和序列号标记),Molex连接器的四个小开口面朝下。然后将MP6/MP6 PP-Flex滑入Molex连接器。夹紧Molex接头,完成两个部件之间的连接。
  3. 制造类似于图15A和15B所示的三个定制的有机玻璃垫片。还准备软木垫以减小尺寸,以补偿微泵和MOLEX连接器的厚度差异。
  4. 制作一个由两个MP6微型泵组成的组件,如图15A(前部)和15B(侧)所示:首先,用双面胶带将有机泵的微泵1的标记侧(MP6-1)粘在有机玻璃隔板上。将软木垫粘贴到第一微型泵的无标记侧。所得到的部件的顺序是:有机玻璃/MP6-1/软木。
  5. 重复这些步骤,微泵2(MP6-2),但相反的顺序,即>,软木垫胶合到标记侧,有机玻璃垫片在未标记的位置。所得到的部件的顺序是:软木/MP6-2/有机玻璃。
  6. 建立一个四MP6微泵组件(图15C),由两个有机玻璃隔板/MP6/软木垫组件组成,通过将第三个(未胶合)的有机玻璃垫片放在中间,从而完成阵列中的零件的顺序是:
    有机玻璃/MP6-1/软木/有机玻璃/软木/MP6-2/有机玻璃。
  7. 所得到的组件可以用双面胶带或夹持器固定在一起,或者用长螺钉穿过钻孔进入有机玻璃垫片。在后一种情况下,务必将螺纹插入到三个间隔件中的一个孔中。
  8. 用铜丝网制作小型法拉第笼(图15F),以消除压电微泵产生的噪音(见注释3)。将小型法拉第笼子搁置在工作台的主地面上。
  9. 完整的组件包括两个或四个MP6微型泵(图15F)。如上所述,抽吸需要四个并联的泵,两个具有独立入口和出口的泵从记录室网格的两侧向海马切片的顶部和底部灌流。
    < BR> < BR> < IMG类=“DoOtStWh”宽度=“100”高度=“156”ALT=“”SRC=“/附着/图像/ 20190926 /201992626432588315.JPG”/>
    图15。组装的微泵阵列和相关组件的不同视图。A和B。两台MP6微型泵并联叠放,使其体积流量相加。每个这样的“三明治”阵列由三个定制的有机玻璃面板,其中一个是螺纹,使螺丝可以保持组装在一起。软木垫按尺寸切割,可精确接触不同表面。c.如(a)和(b)所示的两个组件堆叠在一起,构成控制抽吸的泵阵列。请注意Y型连接器和MP-CV阀。D. 26针组装的PI鞋帮断路板被连接到具有较长引脚的26针屏蔽堆叠头上,以便为直接连接在其上的MP6 OEM控制器中的两个控制器提供足够的间隙。e.将六个微型泵连接到pi-cobbler转接板的电缆。F. Mini Faraday笼由铜丝网制成,以允许来自泵的噪音接地。< BR> 注:阿尔法导线多导体电缆有自己的颜色方案,与图2b、2c、9和11的颜色方案无关。>
    < BR>
第二部分:啮齿动物海马切片制备
< BR>
  1. 准备解剖和记录溶液:准备两份10X ACSF储备溶液,一份用于解剖(解剖10X ACSF),一份用于记录(记录10X ACSF)。将两种10x ACSF储备溶液保持在4°C,直到试验当天。
  2. 在实验当天,准备1份解剖ACSF和1份记录ACSF。
  3. 在室温下用95%o2/5%co2将两种溶液氧化至少20分钟。
  4. 降低切片温度有助于防止缺血损伤(见注2)。为了在不冻结缓冲液的情况下将室温1X解剖ACSF缓冲液快速冷却至适当的低温,将其放入-70°C的冰箱中冷冻45分钟。如果形成冰晶,用小勺子或筛子将其取出,以避免与脑组织接触,并避免对脑组织造成可能的损伤。从冷冻室取出后,继续在4℃下用95%o2/5%co2给夹层acsf充氧。
  5. 准备接口电生理仪:加热接口室水套,使记录室的温度为31.5°C。将250-500毫升的1X记录ACSF放入32°C水浴中,同时用95%O2/5%CO2进行氧化,预热。将溶液预热到略高于记录室温度的温度的目的是防止溶液从热套中通过后再次加热时在管内和网下形成气泡。以500μl/min的流速,用1个记录ACSF的溶液开始灌注。
  6. 在麻醉室内用异氟醚汽化器对动物进行深度麻醉:100%o2持续2分钟,然后100%o2+1.5%异氟醚持续1分钟,然后100%o2+2.5%异氟醚持续2分钟。
  7. 当动物被深麻醉时,用小动物断头台断头。将斩首的头部浸入一个金属外科托盘中,该托盘位于冰箱中的冰上,并装满含氧的冰冷1X解剖ACSF。
    注:动物断头后,脑片的制作应在10分钟以内完成。 >
  8. 快速清除脑部:在头皮上用单刃剃刀刀片切开颅骨。
  9. 用一把锋利的尖刀沿着头骨内侧的外科剪刀,沿着从大孔到前额的矢状缝进行切割。
  10. 用手术剪刀在颅骨颞侧的大孔处各剪一次,然后沿冠状缝横切额骨。用咬骨钳小心地撬开颅骨,露出大脑。
  11. 将头盖骨倒置,用抹刀切断将大脑固定在头盖骨上的颅神经,让大脑落入一个50毫升的烧杯中,烧杯内装有冰镇氧合1次ACSF解剖。
  12. 使用冰箱,将烧杯和大脑一起转移到4℃的冷藏室。
  13. 分离海马体:用90毫米Pyrex培养皿覆盖湿润的滤纸作为解剖平台。
  14. 将冷冻后的大脑放在平台上,沿纵裂平分。将右半脑放在一个50毫升的烧杯中,烧杯内装有1个冰冷氧合的解剖ACSF。
  15. 为了分离左半脑的海马体,用一个扁平的刮刀切断中脑和脑干的残余部分。挖出丘脑,露出海马体的腹面,海马体嵌套在皮层内,清晰可见,呈弧形结构。用抹刀从皮层切断海马的间隔和颞叶连接。用填充的塑料巴斯德吸管,轻轻地将几滴冰冷氧合1X切割ACSF挤到侧脑室的口袋中,以区分伞的边界。
  16. 轻轻地将一支细刷子插入侧脑室,刷上冰凉含氧的1x解剖ACSF,轻轻地将海马体从周围皮质向外滚动。用平铲将剥下的海马体从皮质上割下,将其分离出来。将左侧海马体放置在一个20毫升的烧杯中,其中有一个冰冷氧合的1x解剖ACSF。
  17. 对右海马重复前面的步骤。
  18. 切片:切片阶段应提前准备,可多次重复使用。它由一块直径与McIlwain剪纸机塑料盘相等的圆形薄软木垫组成,并用平行的双面胶带粘在上面。McIlwain组织切碎器解剖平台的夹钳可以调整以匹配切片阶段的厚度(图16c)。
  19. 将一张滤纸放在切片台上,并用冷冻氧合1X解剖ACSF湿润。
  20. 用细刷子(或扁平的一面呈直角弯曲的抹刀)轻轻地将海马体从烧杯底部提起。
    < BR> 图16。显示左侧大鼠海马在McIlwain组织切碎器解剖平台上正确定位的照片(详情请参阅正文)
    < BR>
  21. 将分离出的海马体放在切片阶段,顶部朝上(背侧包含肺泡)。通常可以用光纤的斜射光观察到这一侧肺泡表面的条纹。用刷子轻轻地拉直海马体的底面,使其完全平放在湿润的滤纸上。
  22. 只有背侧海马体会从两侧被切开。为了固定海马体,可以用昆虫别针将腹侧海马体(较厚的一端)固定在切片阶段的软木塞上(图16a)。
  23. 通过McIlwain组织切碎器解剖平台(图16c)的夹钳滑动切片阶段和固定的海马体。
  24. 将一张滤纸放在切片台上,并用冷冻氧合1X解剖ACSF湿润。
  25. 用细刷子(或扁平的一面呈直角弯曲的抹刀)轻轻地将海马体从烧杯底部提起。
    < BR> < IMG类=“DoOtStWh”宽度=“315”高度=“100”ALT=“”SRC=“/附/图像/ 20190926 / 209926264401Y954 8. JPG”/> NBS
    图16。显示左侧大鼠海马在McIlwain组织切碎器解剖平台上正确定位的照片(详情请参阅正文)
    < BR>
  26. 将孤立海马放在切片阶段,它的顶侧(背侧包含有乳头)。通常可以用光纤的斜射光观察到这一侧肺泡表面的条纹。用刷子轻轻地拉直海马体的底面,使其完全平放在湿润的滤纸上。
  27. 只有背侧海马体会从两侧被切开。为了固定海马体,可以用昆虫针脚将腹侧海马(较厚端)固定在切片阶段的软木上(图16A)。
  28. 通过McIlwain组织切碎器解剖平台(图16c)的夹钳滑动切片阶段和固定的海马体。
  29. 在平台上旋转切片平台,使背海马相对于剃刀刀片正确定向:刀片应与背侧海马的横轴线形成15°角(图16B,16C)。当使用该轴的角度为15°至30°时(Alger 等, >,1984),兴奋性通路得到更好的保留。一般来说,在这个方向上,Alvess条纹将趋向于平行于刀片运行。如果使用左半脑的海马体,从上方看,它将看起来像一个直立的“U”,背部海马体在右侧,腹侧海马体(固定)在左侧(图16a-16c)。要获得正确的切割角度,逆时针旋转切片阶段15°。同样,如果使用右侧海马体,它将像一个倒“U”,并且该阶段顺时针旋转15°。
  30. 用ACSF湿润刀片,通过海马体降低刀片制作切片。当叶片上升时,每一片都应该粘在叶片的湿表面上。前3个(最背面)完整的切片通常被丢弃。根据动物的年龄,可从大鼠身上获得6至8片450 mm厚的切片(图16b),从小鼠身上获得4-5片这样的切片。叶片的速度和强度应调整,以便切片被轻轻地切割,但足够快,以避免未切割的海马附着在上升叶片上。刀片不应猛击在切割台的表面上;理想情况下,它只应在切碎台上的湿滤纸表面凹陷。
  31. 使用湿润的细貂皮刷快速从刀片上移除切片。在制作每个切片后,立即将其转移到充有冷氧1X解剖ACSF的试管中。在搬运过程中,小心避免拉伸或过度弯曲切片。一种方法是用刷冷的含氧1X解剖ACSF预处理的叶片,用温和的滚动运动来选择切片。刷子上的一些ACSF水分也会粘在刀刃上,帮助下一片在海马体被切掉后粘在刀刃上。
  32. 制作切片后,立即将其转移至接口记录室,以恢复至少2 h。切片应放置在气液界面的网格上,在其周围形成一个小弯月面(图17a、17b和17c)。在500μl/min的流速下,从顶部和底部持续超负荷使用(非循环)1X记录ACSF。

注:还请参考以下视频文章,提供有关长期记录用急性海马切片制备的详细信息:Villers and RIS(2013);Shetty>等人。>(2015)。>
< BR>
图17。显示大鼠海马切片在奥斯陆式界面记录室中正确定位的照片。a.切片位于靠近录音槽壁的网格上。这个录音室有两个这样的录音浴缸,两个都被一个可移动的凸盖覆盖,在切片上方形成一个加湿的富氧大气。顶棚有一个三角形的开口,允许从每个切片单独记录(而另一个浴槽中的切片仍被覆盖)。每一片都不断地被过量使用,ACSF通过流入管流入镀液,然后通过流出管道(穿过流入)流出,流入管道通向抽吸井(抽吸由黄色注射器针头提供)。在界面处,切片周围形成一个小的半月板。在槽壁周围形成一个较大的弯月面。
< BR> 第三部分:循环模式下寡核苷酸在切片中的应用 < BR> 在ODN实验中,在界面模式下从分离中恢复后,增加浴水平,使切片完全浸没,并允许含有20μm寡核苷酸的多余物再循环(5ml总体积,5ml/m in,持续30min)。为此,请执行以下步骤:

  1. 在图14的顶部Arduino(图2A中的“微控制器1”)上复制并加载图18的程序。将“延迟”值更改为“1800000”,保存新程序并重新加载到顶部Arduino。通过WinLTP程序激活ADC的数字输出后,顶部Arduino将开启所有六个微型泵30分钟。
  2. 加载Arduino脉冲宽度调制库(参见链接),复制并加载图14底部Arduino的图19程序(图2中的“微控制器2”)。将“Int32时钟频率”值更改为“200”,保存新程序并重新加载到底部的Arduino。通过WinLTP程序激活ADC的数字输出后,底部的两个泵将向记录室的入口提供5 ml/min的流速(图20)(一旦启动,吸入口的四个OUFLOW泵将始终以最大流速运行)。
  3. 关闭蠕动泵,同时关闭真空抽吸。
    < BR>
    图18。Arduino程序,可同时激活所有6个MP6原始设备制造商,并在指定时间内保持其活动状态。当ADC的一个数字输出提示时,程序允许同时打开所有6个MP6微型泵,而ADC又由WinLTP程序的“Slow0 Perfusion Change”功能控制。通过改变“延迟”值(以黄色突出显示),可以对切片进行不同间隔的灌注。在这个特殊的例子中,泵将保持开启1800000 ms(即,30分钟)。
    < BR>
  4. 使用spst按钮主开关打开压电泵电路。
  5. 使用三通阀在主储液罐的蠕动泵抽吸溶液(通常是用夹子夹在水浴内的250毫升锥形烧瓶)和水浴入口的两个微型泵组件之间手动切换,它应该是从含有ODN循环溶液的15ml锥形管中提取溶液(与主储液罐在同一水浴中)。
  6. 使用顺序协议脚本和自动灌注控制启动WinLTP程序(Anderson等人,2012年;另见WinLTP软件手册第10章(Anderson,2018年)。< BR> 注:自动灌注控制目前仅由国家仪器M或X系列ADC板支持。用缓慢灌注改变将一条灌注线控制到一个细胞外切片室。> BR/> < BR> < IMG类=“DoOtStWh”宽度=“100”高度=“122”ALT=“”Src=“/ /附着/图像/ 20190926 / 2099262600、437、2635、JPG”/>BR/> 图19。执行脉冲宽度调制(PWM)的Arduino程序。程序允许从MP6 OEM产生的信号的频率从默认值改变,以使MP6微泵流量从其最大值(5.5 mL/min)减少。通过更改“Int32_t ClockFrequency”(黄色突出显示)的值,可以更改OEM信号的频率。当前值“Tn32”,CcCurthFiels=<Stase=“背景颜色:αFFE500;>75</SPAN>”,其流量约为2.5 mL/min(见图20)。 注:图书馆下载链接:< HRFF=“http://CODE.GooGeL.CO/Soviv/P/ARDUNIO-PWM频率/图书馆/下载”目标=“空白”>http://COD.谷歌.COM/GuiVi/P/ARDUINO PWM频率库/下载 < BR> < IMG类=“DoOtStWh”宽度=“126”高度=“100”ALT=“”SRC=“/附着/图像/ 20190926 /2019926264604Y7670.JPG”/>
    图20。通过脉冲宽度调制(PWM)改变单个MP6微泵的流量。< /强>通过为“INT32×T时钟频率”(图的X轴)插入不同的值,流速(Y轴)可以从接口室记录所需的每分钟几百微升(插入中所示)的低速率改变,最大流量为5.5 mL/min。
    < BR>

注:从国家仪器M-或X系列板的数字输出连接到ARDUINO 1,它将激活两个独立的微型泵组成的组件,控制进水口到浴缸,以及控制抽吸的多泵阵列。确保吸液器输出端的管将溶液引回15毫升锥形管,以完成再循环回路。>

  1.  ;如有必要,调整腔室吸入井中吸入管的高度,以提高熔池液位并将切片完全浸没。
  2. 在浸没模式下再循环30分钟并开始记录后,通过将吸气管降低到界面水平,并通过改变从200到10的“32位时钟频率”值,将剩余流量降至0.5 mL/min,降低含ODN溶液的浴位。再次加载图20的程序到Arduino Uno的底部。
  3. 将刺激和记录电极放置在感兴趣的海马层并开始电生理记录。
  4. 为了研究长期突触可塑性持续数小时的实验,记录场EPSPS(FEPSPS)与玻璃细胞外记录电极(2-5 MΩ)放置在CA1层放射状物,与同心双极刺激电极放置在CA3或CA1的任何一侧。如果初步分析显示FEPSP峰值阈值为2 mV,则从研究切片中排除。确认两个刺激通路之间的独立性,因为在两个通路之间没有配对脉冲促进。将基线FEPSP设置为尖峰阈值的25%,并通过在0.033 Hz处向每个路径递送刺激来监测它。通过强HFS诱导LTP,例如,标准两个100 HZ-1S破伤风列车,间隔20 s,这是优化的,以产生相对快速的蛋白质合成起始依赖于LTP(TSOKAS 等.,2005;TSOKAS 等。,2016,Hsieh 等。,2017)。大鼠海马脑片在破伤风阈值的70%处递送破伤风(Tsokas等2005;Hsieh 等2017),小鼠切片使用25%的尖峰阈值(Tsokas . > 2016)。可以使用WiLTP数据采集程序(乔林和CulrGrink,2007)在PC上分析FEPSP的上升的最大斜率。

笔记

  1. 界面与浸没腔的比较:APKC抑制剂ZIP的氧合作用及其对突触传递的影响 大多数研究海马脑片的长期突触可塑性,采用奥斯陆型接口录音室,而不是淹没室,因为前者被认为能提供良好的氧合和保存正常的突触功能(Khurana和李,2013)。我们用电化学溶解氧探针(阿特拉斯科学KIT-106;锌阳极,银阴极,15%四硼酸钠/ 15%氯化钠电解液)测试了这个假设;聚乙烯膜)测量奥斯陆型接口记录室和同一个腔室中的溶解氧(DO)浓度,流速为2~15 mL/min。  ;按照制造商的说明,使用蒸馏水和环境空气校准溶解氧探针。通过在室温(23.5°c)下测定acsf(14000μs/cm)中的溶解氧,进一步验证了仪器的准确性。平均值(8.1±0.01 mg/L;n=5)与以前报道的不同温度和盐度条件下的大气溶解氧平衡数据(RaTKE等, >,1998)一致。  ;然后,在31.5°C下,从医疗气缸或100%氧气(均来自TW Smith)中的空气通过奥斯陆式室水套的气体分散环(或陶瓷曝气器)鼓泡,并在接口条件下,在记录室内测量溶解氧浓度,探针夹在微操作器,并保持在网格表面上方几毫米(通常形成围绕切片的弯月面)。因此,用ACSF湿润的探针尖端产生了氧饱和度与浓度的标准曲线(21%O2;31.1±0.8mg/L,100%O2;n=5;线性拟合通过0,r2=0.99)。当95%o2-5%co2气泡通过水套时,界面薄片上方的氧气浓度为29.6±0.7 mg/L(95.1±2.3%饱和;n=5)。
     ;然后,奥斯陆型舱被转换成一个潜水舱:提高吸力以增加记录槽内的过量盐水平,关闭通过水套中曝气器的充氧,并增加流速以获得2、5、10和15 ml/min时的氧浓度/饱和度测量值。在潜水条件下,在切片实际实验位置的正上方,获得以下氧浓度(mg/L)和饱和度百分比测量值(n's=4)–2 ml/min:19.6±0.9(62.5±2.8%);5 ml/min:22.7±1.4(72.4±4.6%);10 ml/min:25.5±1.7(81.6±5.7%);15ml/min:26.9±1.9(86.3±6.2%)。这些测量值与在其他地方描述的不同类型的浸没室的值一致,使用碳纤维微电极、光学传感器、极谱单元(即、安培克拉克电极)或比色化学(靛蓝胭脂红)方法(Ha JoS等2009);Dondzillo等人,2015年;Weng等人,2018年)。 nbsp;这些结果表明,浸没室(例如,见Volk等人,2013年)的过量使用中的氧饱和度大大低于奥斯陆式界面室中包裹切片的增湿大气中的氧饱和度,特别是在通常使用的流速下(对于流速为72.4±4.6%的浸没室而言5ml/min,与奥斯陆型接口室的95.1±2.3%相比)。在制备后的水下条件下(如VoLK等2013),较低的氧饱和度对切片健康的影响可能进一步加剧。 &我们接着问,在界面室和水下室获得的氧合的差异是否对突触生理有生理影响。我们特别讨论了小室设计是否可以解释非典型pkc抑制剂zip对基底突触传递影响的差异。而在所有已发表的研究中,一致的ZIP逆转LTP(参见,例如,LimeEm>等人,>,2002;Sajikumar 等.,2005;Pastalkova 等人, >,2006),在这些研究中,PKM ZZ抑制剂的作用对增强的突触通路具有特异性,而对基底没有影响,未强直化的突触通路。该抑制剂对增强和非基础突触传递的特异性与应用于LTP和基础突触传递的结果相同,记录在<(Pastalkova 等人, >,2006;Mordro Nal[Em>et al.,2010)。然而,一项研究报道ZIP对晚期LTP和基线突触传递均有影响(Vok等。>,2013)。然而,这项研究是不寻常的,因为晚ltp记录和基底突触传递被记录在海马脑片中,海马脑片被保存在一个水下小室中。&因此,我们直接比较了ZIP对界面和潜水室记录的海马脑片LTP和基线突触传递的影响。为了使所有其他参数尽可能保持恒定,接口室切片在其记录发生的同一个腔室中恢复;而潜水室切片放在接口室中,然后,通过增加ACSF溶液的流速和槽位,立即将其转换为浸没室,然后让切片再恢复90分钟(两个实验中两组切片的总恢复时间保持相等)。
     ;如图21所示,在恢复并记录在界面室的切片中,ZIP特异性地逆转了破伤风途径中晚期LTP的维持,而不影响未经破伤风途径中的基础神经传递。相反,在浸没条件下记录的切片中,ZIP会逆转LTP的维持,最终低于基线,并且对一个独立的未经坦桑尼亚化的路径也有影响。ZIP对未经tanized/基础通路的影响与Volk等人(2013年)所见类似。  ;这种差异的一个合理解释是,有一个非典型pkc库参与了缺氧的神经保护(见tian等人,2008),它与维持晚期ltp的pkmζ库是分开的。由于界面室的氧合条件优于水下切片,ZIP对水下室基底神经传递的影响可能反映了抑制剂对相对缺氧诱导的非典型PKC神经保护作用的影响。切片条件(Tian等人,2008年)。另一种可能的情况是,潜入室的低氧条件可能在未经鞣化的突触中诱导了低氧LTP,使它们对ZIP敏感。重要的是,如上所述,ZIP对增强而非基底突触传递的特异性在海马穿通通路齿状回输入(Pastalkova等人,2006年)和海马CA3-CA1(Madroñal等人,2010年)的活体内可见。以及第4层初级视觉皮层(Cooke和Bear,2010年)。因此,高氧接口室,而不是低氧淹没室似乎产生了突触传递和可塑性的条件,更能反映体内的条件。  ;除了较低的氧合条件外,另一个可能导致Volk等人(2013年)中未经鞣化途径ZIP的人为反应的因素是用于诱发晚期LTP的条件刺激强度。volk等(2013)方案的强度是epsp反应最大值的75%,适用于大鼠schaffer侧支/连合-ca1突触刺激,但远高于通常用于诱导小鼠海马脑片ltp的强度。其通常介于峰值阈值的25%之间,如Tsokas等人(2016年)和Abel等人(1997年)、Nguyen等人(2000年)、Barco等人(2002年)、Opazo等人(2003年)、Selcher等人(2003年)的50%之间,Kelleher等人(2004年),Costa Mattioli等人(2005年),Antion等人(2008年),Pavlowsky and Alarcon(2012年),Gruart等人(2012年),Li等人(2017年),Puzzo等人(2017年),Amorim等人(2018年)。Volk等人(2013年)的这种强重复性突触刺激可能进一步损害了神经元的健康,从而导致未经鞣化途径中的EPSP人为降低到基线以下,以响应ZIP。
    < BR>
    图21。脑切片在界面室和潜水室进行的晚期ltp实验。A.ZIP(5μm)用于晚期LTP逆转增强的维持阶段,对未经鞣化的途径无影响(来自Tsokas等人,2016;n=5)。b.相反,在界面室中恢复的切片(n=3)中(与a中切片的时间相等),然后通过增加acsf的浴位和流速将其转换为浸没室,5μm zip不仅逆转了tetanized通路中的ltp,而且降低了非tanized通路中的基底突触传递。这种差异可能是由于接口室的优越氧合条件所致(奥斯陆型接口室的氧饱和度为95.1±2.3%,而典型流速为5 ml/min的潜水室的氧饱和度为72.4±4.6%)。
    < BR>
  2. 兴奋毒性保护
    为了防止切片制作过程中的兴奋毒性,我们建议使用冰冷高镁/低钙ACSF溶液,并在4°C的冷室内进行切片制备程序。
    &降低温度会降低细胞的代谢率,从而降低其能量消耗,使细胞在切片制备过程中能够在缺血状态下存活。此外,我们建议在解剖过程中使用一种特殊的非生理性ACSF(解剖ACSF),其中含有10毫米镁和0.5毫米钙。这两种偏离生理性脑脊液的行为都可以降低兴奋性毒性(Feig和Lipton,1990;Sacktor等人,1993;Wang和Kass,1997;Ting等人,2018)。
  3. MP6微型泵噪音
    系统中有两个噪声源:(a)原始设备制造商的电信号产生的噪声,和(b)由于泵执行机构的离子ACSF振动而在溶液中产生的噪声。这些噪声源可通过以下方法完全消音:(a)为电路制造金属外壳,为泵“三明治”阵列制造微型法拉第笼,(b)使再循环的ACSF在流入槽和吸入口处接地,使用T形油管接头,将氯化银电线连接至钻机主接地。
  4. 被动止回阀MP Cv
    为了消除由于泵入口和出口之间的压差(例如,由于记录室的高度和ACSF储液罐或连接管的高度之间的差异)导致的ACSF的潜在回流,使用Bartels Mikrotechnik(MP-Cv)制造的不锈钢被动式止回阀。影响微泵容积流量的阀门应设置在微泵和出水池之间。图15c显示了四个这样的MP-Cv止回阀,它们连接到控制接口记录室吸力的四个MP6微型泵阵列上。
  5. 压电泵系统的金属外壳
    出于安全原因(防止来自原始设备制造商的电击),以及消除原始设备制造商产生的噪音和保护电路免受损坏,图14中的完整电路应放置在接地的金属外壳内。外壳应有一个用于连接微型泵组件的分接板带状电缆的开口,以及用于连接顶部Arduino和ADC板的电源电缆、Arduinos的两条USB电缆、电路主开关和BNC电缆的开口。
  6. 混合持久性/压电泵布置
    此处描述的再循环装置的一种简化的替代方案是仅使用压电泵来抽吸再循环ACSF,以及蠕动泵以向腔室提供流入。在这种布置中,具有双通道泵头的蠕动泵将控制流入(可选地,如果接口室支持两个独立的切片浴),则可以使用四通道泵头。一个压电阵列(或两个,取决于由接口腔室支撑的浴的数量)将控制吸气器,每个阵列由四个MP6微泵组成,所述微泵由电路的吸力部分(即,前四个OEM)以最大流速驱动。
  7. 在切换解决方案之前冲洗灌注系统的死体积
    当95% O<2<2/次>->0>2>2/亚>曝气ACSF停留在聚乙烯或Tygon管上很长时间后,被灌注到切片上,使其脱气并变成低氧。二氧化碳的损失也改变了ACSF标准碳酸氢盐缓冲液的pH值。因此,在切换解决方案之前,“预冲洗”灌注系统中的死体积是很重要的。 在TSOKAS 等< (2016)中,两个手动三通阀串联允许通过将上游三通阀从怠速蠕动泵(OFF)切换到怠速MP6微泵(ON),预先冲洗流入的MP6微泵组件的管中的死体积。切换下游三通阀,使微泵流出的液体直接流入废物(开),而不是进入切片室(关)。在这种配置中,将微型泵打开10秒钟,然后将其关闭,使死区容积得以清除。按照这个程序,首先将下游阀的出口转为废物,然后再打开MP6微泵,恢复灌注系统的运行。 WiLTP 2.30有四个灌注管路控制(慢0、慢1、快、快),可用于控制压电步进器和夹管阀,以自动改变浴灌注。有关如何将这些功能与商用夹管阀系统一起用于切片自动预冲洗灌注的出色讨论,请参阅WinLTP 2.30手册(Anderson,2018)第10章。
  8. MP6微型泵很敏感,如果用注射器加压,液体会被强行注入,会严重损坏。如果微型泵在最初开启时无法抽吸液体(通常是由于在先前使用过程中未完全清除其中的液体),最好将MP6的入口连接到垂直放置的20毫升注射筒顶端的适当管道上(即即,无柱塞,且其尖端指向地面)。打开微型泵,将几毫升水注入注射器的筒中,并允许重力帮助微泵振动压电隔膜清除泵的腔室。

食谱

  1. 10x原料解剖ACSF缓冲液(保持在4°C)(表1)
    < BR> 表1。制备10x原料解剖ACSF缓冲液的配方(保持在4°C)
    <正文> <表> < BR>
  2. 10x库存记录ACSF缓冲液(保持在4°C)(表2)
    < BR> 表2。制备10x库存记录ACSF缓冲液的配方(保持在4°C)
  3. 试剂
    库存浓度(10倍)
    1L
    氯化钠< BR> 125毫米<BR/> 73.20克
    KCl<Br/> 2.5毫米<BR/> 1.86 g<Br/>
    nah2po4
    1.25毫米
    1.50 g<Br/>
    nahco3
    26毫米<BR/> 21.84克
    <正文> <表> < BR>
  4. 1x解剖ACSF缓冲液(表3)
    < BR> 表3。制备1x解剖ACSF缓冲液的配方(实验当天制备)
  5. 试剂
    库存浓度(10倍)
    1L
    氯化钠< BR> 118毫米<BR/> 69.00克
    KCl<Br/> 3.5毫米<BR/> 2.61 g<Br/>
    nah2po4
    1.25毫米
    1.50 g<Br/>
    nahco3
    24毫米<BR/> 20.15克
    <正文> <表> < BR>
  6. 1x记录ACSF缓冲器(表4)
    < BR> 表4。制备1X记录ACSF缓冲液的配方(实验当天制备)
  7. 试剂
    工作浓度(1x)
    1L
    cacl2
    0.5毫米<BR/> 0.5毫升1毫升cacl2溶液
    mgcl2
    10毫米<BR/> 10毫升1毫克氯化镁2溶液
    d-(+)-葡萄糖
    11毫米<BR/> 1.98 g<Br/>
    10倍解剖ACSF
    < BR> 100毫升

    < BR> 889.5毫升
    <正文> <表>

    致谢

    这种压电微泵再循环系统和ODN输送协议最初在Tsokas等人(2016)中使用。这项工作得到了NIH拨款r21ns091830和r01mh099128(aaf)和r37mh057068、r01mh115304、r01mh53576、r01da034970以及轻型战斗机信托基金(tcs)的支持。我们感谢Randy Andronica提供的技术援助。作者要感谢davide b.r.bianchi(马萨诸塞州波士顿大学)和zoe b.ravera(纽约州纽约菲尔德斯顿伦理文化学校)基于该协议构建原型,以测试其准确性。Panayiotis Tsokas是亚力山大·S·奥纳西斯公益基金会学者。

    相互竞争的利益

    作者声明不存在任何利益冲突。

    伦理学

    动物实验:Tsokas等人(2016)的实验严格按照国家卫生研究院《实验动物护理和使用指南》的建议进行。所有动物均按照批准的机构动物护理和使用委员会(IACUC)协议(Suny Downstate医疗中心的11-10274、12-10298、13-10363或纽约大学的15-1459)处理。这些方案得到了Suny Downstate医疗中心(动物福利保证编号:A3260-01)和纽约大学(动物福利保证编号:A3317-01)的机构动物护理和使用委员会的批准。所有手术均在氯胺酮和右美托咪定、异氟醚或戊巴比妥钠麻醉下进行,并尽一切努力减少痛苦。

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Copyright Tsokas et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Tsokas, P., Rivard, B., Hsieh, C., Cottrell, J. E., Fenton, A. A. and Sacktor, T. C. (2019). Antisense Oligodeoxynucleotide Perfusion Blocks Gene Expression of Synaptic Plasticity-related Proteins without Inducing Compensation in Hippocampal Slices. Bio-protocol 9(19): e3387. DOI: 10.21769/BioProtoc.3387.
  2. Tsokas, P., Hsieh, C., Yao, Y., Lesburguères, E., Wallace, E. J. C., Tcherepanov, A., Jothianandan, D., Hartley, B. R., Pan, L., Rivard, B., Farese, R. V., Sajan, M. P., Bergold, P. J., Hernández, A. I., Cottrell, J. E., Shouval, H. Z., Fenton, A. A. and Sacktor, T. C. (2016). Compensation for PKMζ in long-term potentiation and spatial long-term memory in mutant mice. Elife 5. pii: e14846.
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