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Electrophysiological Recordings of Evoked End-Plate Potential on Murine Neuro-muscular Synapse Preparations
电生理记录小鼠神经肌肉突触制备物中诱发的终板电位   

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PLOS Pathogens
Aug 2017

Abstract

Neuromuscular junction (NMJ) is the specialized chemical synapse that mediates the transmission of the electrical impulse running along motor neuron axons to skeletal muscle fibers. NMJ is the best characterized chemical synapse and its study along many years of research has provided most of the general knowledge of synapse development, structure and functionality.

Electrophysiology is the most accurate experimental procedure to study NMJ physiology and it largely contributed to the elucidation of synaptic transmission basic principles. Many electrophysiological techniques have been developed to study NMJ physiology and physiopathology. In this paper, we describe an ex vivo tissue preparation for electrophysiology that can be applied to investigate nerve-muscle transmission functionality in mice. It is routinely used in our laboratory to study presynaptic neurotoxins, antitoxins, and to monitor NMJ degeneration and regeneration. This is a broadly applicable technique which can also be adopted to investigate alterations of NMJ activity in mouse models of neuromuscular diseases, including peripheral neuropathies, motor neuron disorders and myasthenic syndromes.

Keywords: Neuromuscular junction (神经肌肉接头), Electrophysiology (电生理), Evoked End-Plate Potential (eEPP) (诱发终板电位(eEPP)), Miniature End-Plate Potential (mEPP) (微型终板电位(mEPP)), Neurotoxins (神经毒素), Regeneration (再生)

Background

Neurotransmission is the physiological process by which neurons transfer information to target cells on a rapid time scale (usually < 1 msec). The structure mediating this communication is the synapse, a specialized structure formed either between neurons (a pre- and a post-synaptic neuron) or between a neuron (presynaptic neuron) and an effector cell (post-synaptic cell). Neuromuscular junction (NMJ) is the chemical synapse enabling communication between motor neuron and skeletal muscle fiber. This is the best characterized synapse and most of the knowledge on maturation, structure and function of synapses derives from its study (Li et al., 2016). At the NMJ, the action potential running along the motor axon invades the nerve terminal (presynaptic bouton) and induces the opening of voltage-gated calcium channels. The ensuing Ca2+ influx in presynaptic nerve terminal triggers (approximately in 0.3 µsec (Kuffler et al., 1984)) the fusion with the presynaptic membrane of about 100 synaptic vesicles from a ready to release pool (10-20% of all vesicles) (Del Castillo and Katz, 1954; Denker and Rizzoli, 2010). Around 1,000 acetylcholine (ACh) molecules per vesicle diffuse in the synaptic cleft (Kuffler and Yoshikami, 1975) and, in about 0.5 msec, bind to the nicotinic ACh Receptors (nAChRs) on the postsynaptic muscle fiber membrane. nAChRs are ionotropic ligand-gated Na+/K+ channels which open upon ACh binding and cause a local depolarizing potential of the postsynaptic membrane (end-plate) by mediating a large inward flux of Na+ (and a smaller outward flow of K+). This local depolarization is named evoked End-Plate Potential (eEPP) (or Evoked Junction Potential). In mice, the resting membrane potential of a skeletal muscle fiber lies around -75 mV and the eEPP has an amplitude of ~15-30 mV (depending on muscle type). When the eEPP amplitude is sufficiently high to reach or overcome action potential threshold, voltage-gated Na+ channels open thus triggering an action potential into the muscle fiber, which ultimately spreads out along the sarcolemma and invades muscle fiber T-tubules. Here, an excitation-contraction molecular machinery transduces this electric signal into the cytosolic release of Ca2+ from the sarcoplasmic reticulum, leading to muscle fiber contraction (Figure 1).


Figure 1. Mechanism of muscle fiber contraction. Acetylcholine (ACh), released by synaptic vesicle (SVs) fusing with the motor axon terminal membrane, binds to post synaptic nicotinic ACh Receptors, ionotropic cation channels that upon binding allow the leakage of cations (Na+ inward, K+ outward), leading to a local depolarization of the sarcolemmatic membrane (eEPP). When depolarization is sufficiently large to overcome the voltage threshold (red dotted line), voltage gated Na+ channels get open and trigger a post synaptic action potential (AP) spreading along the sarcolemma and invading the T-tubules (invagination of the sarcolemma within the muscle fiber). Dihydropyridine (DHP) receptors sense this membrane depolarization and stimulate the opening of Ryanodine Receptors (RyR) on the sarcoplasmic reticulum which release Ca++ into the cytosol eliciting muscle contraction. µ-conotoxin inhibits voltage gated Na+ channels thus allowing to record membrane depolarization due to the opening of the sole nicotinic AChR, i.e., the eEPP.

Random fusion of synaptic vesicles also takes place in the absence of a presynaptic action potential thereby inducing a very small (~0.4 mV) depolarization of the end-plate, which is not sufficient to trigger muscle contraction. This spontaneous activity is called ‘miniature End-Plate Potential’ (mEPP) and, according to the quantal hypothesis, it is generated by the release of a single synaptic vesicle (Katz, 2003).

NMJ is easily accessible to many kinds of experimental manipulation. Since the ‘50s’ electrophysiology applied at the NMJ provided seminal discoveries on basic aspects of synaptic transmission (Augustine and Kasai, 2007). Thereafter, a continuous development in technics and animal models paved the way to sophisticate investigation of pathological alterations occurring at the NMJ in neuromuscular diseases, including myasthenic syndromes and peripheral neuropathies, as well as neuroparalytic syndromes caused by animal (Duchen et al., 1981; Duregotti et al., 2015a) and bacterial toxins (Colasante et al., 2013, Pirazzini et al., 2014).

We describe here a detailed protocol to evaluate NMJ functionality in murine muscle-nerve preparations. The method is based on the intracellular recording of spontaneous mEPPs and nerve-evoked EPPs in muscle fibers of soleus nerve-muscle preparations, thus allowing accurate investigation of NMJ functionality at a single synapse resolution (Tremblay et al., 2017). We have recently used this method to test engineered botulinum neurotoxins and to assay the efficacy of novel putative antitoxins (Pirazzini et al., 2014; Zanetti et al., 2017). In addition, we successfully employed this technique to study NMJ nerve degeneration and to test molecules promoting its regeneration (Duregotti et al., 2015b; Negro et al., 2017; Rigoni and Montecucco, 2017).

This procedure represents a basic technique that can be easily adopted to investigate NMJ activity in mouse models of any neuromuscular diseases, including peripheral neuropathies, motor neuron disorders and myasthenic syndromes.

Materials and Reagents

  1. Silver wire (World Precision Instruments, catalog number: AGW2030 )
  2. 1 ml syringe (CHEMIL s.r.l., Padova, catalog number: S01G25 )
  3. Petri dish 35 mm (any producer is fine)
  4. Petri dish, 35 x 10 mm, coated with Sylgard (Dow Corning, Sylgard® 184 Silicone Elastomer kit)
  5. Flexible needle electrode Microfil (World Precision Instruments, catalog number: MF34G-5 )
  6. Tips for 2 μl micropipette
  7. Tips for 200 μl micropipette
  8. Tips for 1,000 μl micropipette
  9. Glass capillaries for intracellular microelectrodes (length 100 mm, inner diameter 0.86 mm, outer diameter 1.50 mm; Science Products, catalog number: GB150F-10 )
  10. Glass capillaries for stimulating microelectrode (length 100 mm, inner diameter 1.05 mm, outer diameter 1.50 mm; Science Products, catalog number: GB150TF-10 )
  11. Mice of desired strain and age
    Note: We used here plp-GFP C57BL/6J transgenic mice.
  12. Iron(III) chloride (FeCl3) (Sigma-Aldrich, catalog number: 451649 )
  13. Silver chloride (AgCl) (Sigma-Aldrich, catalog number: 204382 )
  14. μ-Conotoxin GIIIB (Alomone, Jerusalem, Israel)
  15. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S5761 )
  16. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
  17. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
  18. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
  19. Magnesium chloride, standard solution 1 M (MgCl2) (Honeywell International, Fluka, catalog number: 63020 )
  20. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C5080 )
  21. Hydrogen chloride (HCl) (Sigma-Aldrich, catalog number: H1758 )
  22. Potassium acetate (CH3COOK) (Sigma-Aldrich, catalog number: P3542 )
  23. Ringer’s solution (see Recipes)
  24. Recording electrode solution (see Recipes)

Equipment

  1. Micropipettes
  2. Volumetric flask (typically 500 ml; any producer is fine)
  3. Electrophysiology setup complete with antivibration table (Newport, USA) (Figure 3)
  4. Stereomicroscope for the electrophysiology setup (Leica Microsystem, model: Leica MZ125 , numeric aperture 0.8 with plan apochromatic objective 1.6x) (#1 in Figure 3)
  5. Hydraulic micromanipulator for intracellular recording electrode (NARISHIGE, model: MHW-103 , Three-axis Water Hydraulic Micromanipulator) (#2 in Figure 3)
  6. Stimulation electrode micromanipulators (Manual Micromanipulator, MÄRZHÄUSER WETZLAR, model: MM 33 ) (#3 in Figure 3)
  7. Faraday cage (#4 in Figure 3, home-made) and stimulator: S88 stimulator (Grass, Warwick, RI, USA) (#5 in Figure 3)
  8. Amplifier: intracellular bridge mode amplifier (Npi electronic, model: BA-01X ) (#6 in Figure 3)
  9. 2 Forceps (Micro Jewelers Forceps, Rudolf Medical, catalog number: RU 4240-05 )
  10. Scissors (Micro Spring Scissors, Rudolf Medical, VANNAS, catalog number: RU 2260-08 )
  11. Scissors (Delicate Surgical Scissors, Rudolf Medical, catalog number: RU 1503-12 )
  12. Dissection microscope (OPTIKA Microscopes, model: SZM-LED2 )
  13. Pipette puller (P-97 Flaming/Brown Micropipette Puller) (Sutter Instruments, model: P-97 )
  14. A/D interface (National Instruments, model: PCI-6221 ) and computer compatible with the software (#7 in Figure 3)
  15. Gas tank 95% O2 with 5% CO2 (any size and any supplier are fine)
  16. Cylinder pressure regulator (Air Liquid, model: HBS 240-1-2 )

Software

  1. Recording: WinEDR free software (Strathclyde University, Glasgow, Scotland, UK)
  2. Analysis: Clampfit (Molecular Devices, Sunny-vale, CA, USA)

Procedure

  1. Solutions and setup preparation
    1. Pull the microelectrode for intracellular recording from filamented borosilicate glass (outer diameter: 1.5 mm; inner diameter 0.86 mm) and the microelectrode for nerve stimulation from filamented borosilicate glass (outer diameter: 1.5 mm; inner diameter 1.05 mm) using a pipette puller. Many electrodes can be pulled and stored at RT until use.
      Note: The exact settings for pulling intracellular or stimulating microelectrodes may vary according to the glass (borosilicate vs. aluminosilicate), thickness of glass capillary wall, glass capillaries manufacturer and batch, puller heating lamina dimension, material and age, pulling velocity, time, steps, heat of each step, and also environmental conditions (humidity). These parameters are particularly relevant for intracellular microelectrodes and must be accurately set up until electrode resistance is optimal (see below). This requires a careful understanding of glass microelectrode behavior under heating conditions, and a clear understanding of puller function and response when settings are adjusted to new values. It is advisable to pull many capillaries with different puller settings until correct parameters are defined. Resistance can be measured by utilizing a specific intracellular amplifier circuit which displays the measured resistance according to Ohm’s law. A desirable resistance should be between 10 and 30 MΩ, values which substantially correspond to very small tips, in the order of 0.5 micrometer diameter (see Figure 2). Tip size of stimulating electrode is not crucial as it can be adjusted to nerve diameter by breaking it with gentle touches toward the bottom of the recording chamber (see Figure 2A).
    2. Buffer the Ringer’s solution (Recipe 1) to pH 7.4 by bubbling with 95% O2, 5% CO2 for at least 15 min.
      Note: About 10 ml of solution is needed for one standard experiment.
    3. Prepare silver chloride wire. Formation of AgCl is achieved by chemical oxidation of the Ag-coated electrode in 10 mM FeCl3/HCl solution. Immerse the electrode in the solution for 10 sec.
      Note: This step should be periodically repeated (about every 2-3 weeks) due to the scraping of AgCl layer upon repeated insertion into glass microelectrode.
    4. Back-fill recording electrodes with recording electrode solution (Recipe 2). This step is carried out in two sequential steps:
      1. Prefill the electrode by heating (at mid-length) with a lighter for some seconds and by rapidly bathing the bottom into the solution. Leave the prefilled electrode horizontal to allow the solution flowing upwards to the tip by capillarity (Figure 2B). This takes about 30 min;
      2. Using 1 ml syringe and Electrode Microfil capillaries, add recording electrode solution until the electrode is almost filled (see Figure 2C).
        Note: Avoid the injection of air bubbles during electrode filling.


        Figure 2. Stimulation and recording electrodes. A. Stimulation electrode. Pre-filled (B) and filled (C) recording electrode. Arrows indicate the level of solution filling.

    5. Switch on the components of the electrophysiological setup (Figure 3).


      Figure 3. Electrophysiological setup components. 1) Stereomicroscope dedicated to the electrophysiology set up; 2) Intracellular electrode micromanipulator; 3) Stimulation electrode micromanipulator; 4) Faraday cage; 5) Stimulator; 6) Amplifier; and 7) Computer with WinEDR free software.

    6. Connect recording (filled) and stimulation electrodes to their electrode-holders. Place a Petri dish filled with Ringer’s solution under the microscope and immerse the recording electrode tip into the solution. Switch on the amplifier and test the resistance. Optimal values are between 10 and 30 MΩ.
      Notes:
      1. Electrode resistance is indirectly proportional to tip diameter (the larger the tip, the lower the resistance). This relationship may be altered by experimental issues: i) incomplete filling or obstruction of the tip, due to the formation of salt microcrystals or the presence of micro-air bubbles, tend to increase the resistance. To restore suitable values, hold the electrode tip immersed in the Ringer’s solution for a few minutes: salts should dissolve and resistance decrease. If not, gently rub the tip of the electrode on the bottom of the chamber to cause ‘a controlled damage’ to the tip. Resistance should drop.
      2. A sudden increase of microelectrode resistance is generally due to tip obstruction by tissue debris (connective tissue or adipose tissue). In this case, the tip can be cleaned by passing large amounts of positive or negative currents (many amplifiers have an ‘electrode cleaner function’). Alternatively, rubbing the tip against the bottom of the chamber may also help. If the resistance does not drop and/or the recording trace is noisy, the electrode must be replaced. When resistance is below 10 MΩ or the tip is too wide (it has been damaged), the electrode must also be replaced.
    7. Switch on the stimulator and set the parameters for stimulation: for eEPPs, stimuli of 0.4 msec duration and frequency of 0.5 Hz are suitable.
      Note: These parameters must be optimized to measure the NMJ functionality for two reasons: a) pulse step duration is minimal to prevent an excessive stimulus artifact; b) pulse step frequency is very low to prevent NMJ ‘fatigue’.
    8. Run WinEDR free software.

  2. Dissection of soleus muscle
    1. Euthanize the mouse according to your animal handling protocol.
      Note: All procedures were performed in accordance with the Italian laws and policies (D.L. No. 26, 14th March 2014), with the guidelines established by the European Community Council Directive 2010/63/UE and approved by the veterinary services of the University of Padova (O.P.B.A.-Organismo Preposto al Benessere degli Animali) (protocol 359/2015). All the procedures should be utilized according to the ethical standards of the Institution where experiments are carried out.
    2. Use the scissors to cut the skin all around the ankle. With the mouse belly up cut along the thigh (Figure 4A).
    3. With the aid of a tweezer, skin hind limb muscles. Put the mouse back in supine position and block the paw with a pin. Using a small scissors, cut the Achilles tendon (Figure 4B).
    4. Gently, pull the tendon up and, with Vannas scissors, accompany muscle detachment from the peroneal bone by cutting the connective tissue lateral to the soleus muscle (Figure 4C).
      Notes:
      1. Electrophysiological Recordings of mEPPs and eEPPs can be virtually done in any muscle that is collectible without damaging muscle fibers and with its nerve properly attached. Here we provide the detailed procedure for the soleus muscle that is, in our opinion, the easiest one to harvest.
      2. Cut the connective tissue from the inside to the outside to avoid (soleus) muscle damaging.


      Figure 4. Initial steps for soleus muscle collection. A. Cut skin all around the ankle and along the thigh; B. Cut Achilles tendon; C. Arrows show the cut direction to detach connective tissue avoiding muscle damage.

    5. When the entire soleus muscle is visible, carefully sever the second tendon as close as possible to the knee, using small Vannas scissors (Figure 5A). At this point, the soleus muscle retires towards the Achilles tendon and all the muscles attached to the tendon can be excised without affecting soleus muscle integrity (Figure 5B).
    6. Place muscles in a Petri dish bottomed with sylgard and filled with oxygenated Ringer’s solution. Pin them as shown in Figure 5C, with the soleus muscle toward the operator (black arrow). Place the Petri dish under the stereomicroscope for next steps.
      Note: For dissection, Ca2+ free Ringer’s solution is advisable to prevent muscle contraction or contracture due to mechanical stimulation.


      Figure 5. Collection of lower hind limb muscles. Arrows indicate soleus muscle.

    7. Using forceps, gently lift the soleus muscle by grasping the lower tendon and by cutting the connective tissue that keeps it attached to the gastrocnemius muscle. Continue until the nerve is visible (Figure 6A). The nerve is underneath the soleus and looks like a white wire inserting into the muscle fibers (dotted line in Figures 6A-6C).
    8. Using Vannas scissors, clean up the nerve from the connective tissue (Figure 6B).
      Note: Approximately 0.5 cm of nerve length is needed for optimal stimulation.
    9. When the soleus muscle and its nerve are completely free, cut away the gastrocnemius muscle at the level of Achilles tendon (Figure 6C).


      Figure 6. Isolation and dissection of the soleus with its nerve still associated. Dotted lines spot the nerve; S is the soleus muscle and G is the gastrocnemius muscle.

    10. Pin the soleus to the bottom of the Petri dish via the two tendons (Figure 7A).
      Note: The soleus muscles must be pinned in order to stretch the muscle and facilitate the entry of the recording electrode into the muscle fiber. Avoid excessive stretching which may cause damage to muscle fibers.


      Figure 7. Details of the experimental chamber. A. Muscle positioning into the Petri dish; B. 1) Recording electrode; 2) Stimulation electrode; 3) Ground; 4) Tube for oxygenation; 5) Reference electrode; 6) Soleus muscle.

    11. Gently, clear muscle and nerve from residual tissue debris without damaging the fibers or the nerve.
    12. Rinse muscle one or two times with Ringer’s solution to eliminate connective tissue debris or fur-hairs. Leave the muscle in the Petri dish filled with 2 ml of fresh Ringer’s solution.

  3. Nerve stimulations and EPPs recording
    1. Place the Petri dish under the stereomicroscope of the electrophysiology apparatus and submerge a tube connected to the O2/CO2 gas tank to bubble oxygen in the solution (Figure 7B).
    2. Put the Ground and the Reference of the stimulation electrode in the solution (Figure 7B). Note: Grounding is needed to get rid of electrical noise due to current loops among the apparatuses internal to the Faraday cage (Faraday cage, in turn, is necessary to prevent grid current noise). The Reference is needed to measure the voltage across the muscle fiber membrane, i.e., resting potential and how it varies (mEPPs, eEPPs and action potentials). Membrane potential is measured by comparing the voltage of the recording electrode with that of the Reference, which must be in contact with the extracellular fluid (solution) around the muscle.
    3. Using the micromanipulator, place the stimulation electrode in the solution and aspirate Ringer’s solution (filling approximately half of the electrode volume). Then, approach the electrode to the nerve stump and suck it up into the electrode together with the solution (Figure 8).
      Note: Use a syringe to generate a negative pressure to aspirate the solution and to suck the nerve. The nerve must be completely immersed in the solution to avoid air entry into the electrode and to provide electrical continuity between the electrode and the nerve.


      Figure 8. Positioning of electrodes. A. Recording electrode 1) Soleus muscle, 2) Recording electrode and 3) Nerve. B. Stimulation electrode. 1) Soleus muscle, 2) Recording electrode, 3) Nerve and 4) Recording electrode.

    4. Stimulate the nerve by increasing the voltage intensity until the muscle contracts. For intracellular recordings set the stimulator at a voltage intensity which is 1.5-fold the value necessary to achieve muscle contraction.
      Note: Muscle contraction is generally achieved with a voltage between 5 and 10 V. If contraction is not achieved within 15 V, the nerve can be pushed out and sucked again into the electrode. Alternatively, replace the pins to reduce muscle stretching. If contraction is still not visible, probably the nerve has been injured during the dissection. Avoid voltages above 20 V as muscle contraction may be triggered by direct stimulation of the muscle.
    5. By using the intracellular electrode micromanipulator, place the tip of the recording electrode into the Ringer’s solution of the chamber and turn on the amplifier. Offset the voltage to 0. Move the electrode tip nearby the muscle fibers within the boxed area shown in Figure 9, where the nerve enters the muscle and NMJs are concentrated. When the tip of the electrode approaches the muscle fiber, a slight polarization of the voltage is observed (around -20 mV).
      Note: Even though not strictly necessary, the use of a transgenic mouse with fluorescent reporters in motor neurons or Schwann cells coupled to an epifluorescence stereomicroscope helps in spotting NMJs more accurately.


      Figure 9. Distribution of NMJs. Soleus muscle from C57/BL6/J transgenic mice expressing GFP in Schwann cells under the plp promoter (plp-GFP (Mallon et al., 2002)), imaged with an epifluorescence stereomicroscope. With fluorescent Schwann cells, NMJs can be easily spotted at the end of nerve axons (white circles).

    6. With the micromanipulator move the recording electrode deep toward the muscle. When a muscle fiber is correctly impaled, the voltage (read from the amplifier or the Oscilloscope/Computer interface) suddenly drops to very negative values, corresponding to the resting potential (VRest) of the muscle fiber (generally in between -90 and -60 mV).
      Notes:
      1. The value of the VRest varies from cell to cell and, in some case from the treatment. In this case, recordings are from a skeletal muscle cell where the VRest is between -60 and -90 mV. To obtain comparable eEPPs amplitudes (eEPP amplitude is linearly dependent on VRest (Boyd and Martin, 1955)) VRest is arbitrarily set to -70 mV for standardisation by using current injection commands.
      2. VRest is NEGATIVE. A positive potential likely corresponds to a misplacing of the electrode outside the fiber.
      3. If the resting potential is between -20 and -40 mV, the electrode is not properly inserted into the muscle cell. Amplifiers are generally equipped with the ‘BUZZ function’, a circuit based on oscillations caused by overcompensating the capacitance compensation system, which facilitates electrode entrance into the fiber.
      4. If membrane potential gradually depolarizes (i.e., slowly returns toward 0), muscle fiber membrane may be damaged and leaky or the electrode is getting out. Change fiber.
      5. Damaged fibers will not display the expected membrane potential in any case, thus change fiber.
      6. Prolonged denervation, as in the case of treatment with Botulinum neurotoxins or cut of nerves, increases the resting membrane potential. A common consequence is muscle fibrillation, a spontaneous, random and asynchronous contraction of muscle fibers (Purves and Sakmann, 1974; Moravec and Vyskocil, 2005). Fibrillation can be inhibited by specific drugs (see below).
    7. At this point, mEPPs can be recorded.
      Note1: Upon stimulation, action potentials are elicited and can be recorded. Instead, eEPPs cannot be recorded yet as they are hidden/covered by action potentials.
      Note2: Please consider that action potentials cause muscle contraction that may break the recording electrode or cause its exit from the fiber during the recording.
    8. Add μ-Conotoxin GIIIB to selectively inhibit voltage-gated Na+ channels (Nav1.4 predominantly expressed by muscle) responsible for triggering the post synaptic action potential, thereby stopping muscle contraction. Using a micropipette, add 1 µl of μ-Conotoxin stock solution (1 mM) to the Petri dish, and switch on oxygenation.
    9. After 15 min, stimulate and check muscle contraction. If the muscle does not contract, EPPs can be recorded, otherwise wait additional 5-10 min.
      Note: Deeper muscle fibers may need more time for μ-Conotoxin activity.
    10. Stop oxygenation (solution movement due to bubbling or excessive flowing introduce artifacts to recording) and start the stimulation.
      Note: eEPP amplitude is directly proportional to the amount of neurotransmitter release that in turn is directly proportional to the number of synaptic vesicles released upon stimulation. eEPPs can be therefore used to test the functional status of nerve endings, providing an accurate estimation of presynaptic effect.
    11. Set recording time (at least 30 sec) and record eEPPs in current-clamp mode. Repeat the procedure for a dozen fibers per muscle (if eEPPs are heterogeneous within the same muscle, increase fiber sampling). A typical recording trace is shown in Figure 10, where the stimulation artifact (proportional to the amplitude of the stimulation), the EPP, proportional to nerve terminal activity and the mEPPs can be distinguished.


      Figure 10. Typical trace during acquisition

Data analysis

  1. The WinEDR software provides an EDR file format. Using WinEDR software, export this file as ABF file format compatible with Clampfit software.
    Note: The procedure of data analysis may vary according to the software used for recording and analysis.
  2. Open ABF file with Clampfit software and build a template on a control trace. Defining the shape of eEPPs in a control trace will ‘teach’ to Clampfit software to recognize the shape of eEPPs (or mEPPs) automatically in all other files. Analyse all the files of analyses to be compared with the same template. Clampfit software provides (for each event in the trace) a table with the parameters defining EPP form (start time, end time, peak amplitude, area etc.).
    Note: The general shape of EPP is parabolic. The initial slope is related to the average amplitude of the postsynaptic response to a packet of transmitter (postsynaptic efficacy); the degree of curvature is related to the probability of transmitter release (presynaptic efficacy); the amplitude is related to the number of independent presynaptic release sites in the presynaptic terminal. In general, the ratio between eEPP and mEPP amplitudes is calculated as ‘quantal content’ of eEPP, i.e., the number of vesicles undergone fusion following a presynaptic action potential. Theoretically, a visual comparison of the different curves under different experimental conditions provides insights about what synaptic parameters are altered.

Recipes

  1. Ringer’s solution

    Notes:
    1. Use ddH2O. All the glassware used must be washed with 0.1 M hydrogen chloride to remove any trace of carbonate, and rinsed very well. Once prepared, this solution can be stored at 4 °C, but no longer than 3 months. If deposit or opalescence is present, discard and prepare a fresh solution.
    2. Before the analysis, the Ringer’s solution is saturated with 95% O2, 5% CO2, by aeration for at least 15 min to obtain pH level of 7.4.
  2. Recording electrode solution

Acknowledgments

This work was supported by the University of Padova with ‘Senior Research Grant for young people not employed in the University of Padova’ granted to M. Pirazzini and with a ‘junior fellowship’ to S. Negro and by Fondazione Caritro with ‘Bando 2017 per giovani ricercatori coinvolti in progetti di eccellenza’ granted to G. Zanetti. All the procedures were performed in the laboratory of ‘Neurotoxins, Neuroparalysis and Regeneration’ headed by Prof. Cesare Montecucco at the Department of Biomedical Sciences (University of Padova). The authors declare no competing interests. SN and GZ performed the procedure for soleus muscle preparation with the help of AM. SN and GZ took the pictures to describe all the procedures and prepared the figures. GZ and MP wrote the paper, with the help of SN. All authors reviewed the manuscript and approved the final version.

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  15. Negro, S., Lessi, F., Duregotti, E., Aretini, P., La Ferla, M., Franceschi, S., Menicagli, M., Bergamin, E., Radice, E., Thelen, M., Megighian, A., Pirazzini, M., Mazzanti, C. M., Rigoni, M. and Montecucco, C. (2017). CXCL12α/SDF-1 from perisynaptic Schwann cells promotes regeneration of injured motor axon terminals. EMBO Mol Med 9(8): 1000-1010.
  16. Pirazzini, M., Azarnia Tehran, D., Zanetti, G., Megighian, A., Scorzeto, M., Fillo, S., Shone, C. C., Binz, T., Rossetto, O., Lista, F. and Montecucco, C. (2014). Thioredoxin and its reductase are present on synaptic vesicles, and their inhibition prevents the paralysis induced by botulinum neurotoxins. Cell Rep 8(6): 1870-1878.
  17. Purves, D. and Sakmann, B. (1974). Membrane properties underlying spontaneous activity of denervated muscle fibres. J Physiol 239(1): 125-153.
  18. Rigoni, M. and Montecucco, C. (2017). Animal models for studying motor axon terminal paralysis and recovery. J Neurochem 142 Suppl 2: 122-129.
  19. Tremblay, E., Martineau, E. and Robitaille, R. (2017). Opposite synaptic alterations at the neuromuscular junction in an ALS mouse model: when motor units matter. J Neurosci 37(37): 8901-8918.
  20. Zanetti, G., Sikorra, S., Rummel, A., Krez, N., Duregotti, E., Negro, S., Henke, T., Rossetto, O., Binz, T. and Pirazzini, M. (2017). Botulinum neurotoxin C mutants reveal different effects of syntaxin or SNAP-25 proteolysis on neuromuscular transmission. PLoS Pathog 13(8): e1006567.

简介

神经肌肉接头(NMM)是一种特殊的化学突触,它介导沿着运动神经元轴突运行的电脉冲传输到骨骼肌纤维。 NMJ是最具特色的化学突触,经过多年研究的研究提供了突触发育,结构和功能的大部分常识。

电生理学是研究NMJ生理学的最准确的实验程序,并且主要有助于阐明突触传递基本原理。已经开发了许多电生理学技术来研究NMJ生理学和病理生理学。在本文中,我们描述了一种可应用于研究小鼠神经 - 肌肉传递功能的电生理学组织制备方法。它在我们的实验室常规用于研究突触前神经毒素,抗毒素,并监测NMJ变性和再生。这是一种广泛适用的技术,也可用于研究神经肌肉疾病(包括外周神经病,运动神经元病和肌无力综合征)小鼠模型中NMJ活性的改变。

【背景】神经传递是神经元以快速时间尺度(通常<1毫秒)将信息传递给靶细胞的生理过程。介导这种交流的结构是突触,即神经元(前突触后神经元)或神经元(突触前神经元)和效应细胞(突触后细胞)之间形成的专门结构。神经肌肉接头(NMJ)是使运动神经元和骨骼肌纤维之间能够交流的化学突触。这是最好的特征性突触,大部分关于突触的成熟,结构和功能的知识来源于其研究(Li et al。,2016)。在NMJ,沿着运动轴突运动的动作电位侵入神经末梢(突触前布顿)并诱导电压门控钙通道的开放。随后突触前神经末梢中的Ca 2+流入触发(大约0.3μsec(Kuffler等,1984))与约100个突触囊泡的突触前膜融合从准备释放池(占所有囊泡的10-20%)(Del Castillo和Katz,1954; Denker和Rizzoli,2010)。每个囊泡约1000个乙酰胆碱(ACh)分子在突触间隙中扩散(Kuffler和Yoshikami,1975),并且在大约0.5毫秒内与突触后肌纤维膜上的烟碱ACh受体(nAChR)结合。 nAChR是离子型配体门控Na + / K + +通道,它们在ACh结合后开放,并通过介导大的突触后膜(端板)引起局部去极化潜力Na +的向内通量(以及K +的较小向外流量)。这种局部去极化被称为诱发终板电位(eEPP)(或诱发连接电位)。在小鼠中,骨骼肌纤维的静息膜电位约为-75mV,eEPP的幅度约为15-30mV(取决于肌肉类型)。当eEPP幅度足够高以达到或克服动作电位阈值时,电压门控Na + +通道打开,从而触发动作电位进入肌肉纤维,其最终沿着肌纤维蔓延并侵入肌纤维T管。在这里,一个激发 - 收缩分子机器将这个电信号转导成肌质网Ca2 +的胞质释放,导致肌纤维收缩(图1)。


图1.肌纤维收缩的机制乙酰胆碱(ACh)由与运动轴突终端膜融合的突触小泡(SVs)释放,与突触后烟碱型乙酰胆碱受体(即后绑定的离子型阳离子通道)结合允许阳离子泄漏(Na + +向内,K + +向外),导致肌细胞膜(eEPP)的局部去极化。当去极化足够大以克服电压阈值(红色虚线)时,电压门控Na + +通道开放并触发突触后动作电位(AP)沿着肌膜延伸并侵入T小管(肌纤维内肌膜的内陷)。二氢吡啶(DHP)受体感应该膜去极化并刺激肌质网上Ryanodine受体(RyR)的释放,释放Ca ++进入胞质溶胶引发的肌肉收缩。 μ-芋螺毒素抑制电压门控Na + +通道,从而允许记录由于单一烟碱型AChR即eEPP的开放引起的膜去极化。

突触小泡的随机融合也发生在没有突触前动作电位的情况下,从而诱导端板非常小的(〜0.4mV)去极化,这不足以触发肌肉收缩。这种自发活动被称为“微型终板电位”(mEPP),根据量子假说,它是由释放一个单一的突触小泡所产生的(Katz,2003)。

NMJ很容易进行多种实验操作。 NMJ应用于'50年代'的电生理学提供了关于突触传递基本方面的重要发现(Augustine和Kasai,2007)。此后,技术和动物模型的不断发展为研究NMJ在神经肌肉疾病(包括肌无力综合征和外周神经病)以及由动物引起的神经麻痹综合征发生的病理改变提供了深入研究的方法(Duchen等人, ,1981; Duregotti et al。,2015a)和细菌毒素(Colasante等人,2013,Pirazzini等人 ,2014年)。

我们在这里描述了一个详细的协议来评估小鼠肌肉神经制剂中的NMJ功能。该方法基于细胞内记录比目鱼肌神经 - 肌肉制备物的肌纤维中的自发mEPP和神经诱发的EPP,从而允许在单个突触分辨率下精确研究NMJ功能性(Tremblay等人 ,2017)。我们最近使用这种方法来测试工程化的肉毒杆菌神经毒素并测定新的假定的抗毒素的功效(Pirazzini等人,2014; Zanetti等人,2017)。此外,我们成功地使用这种技术来研究NMJ神经变性并测试促进其再生的分子(Duregotti et al。,2015b; Negro et al。,2017; Rigoni和Montecucco,2017)。

这个程序代表了一个基本的技术,可以很容易地用来调查任何神经肌肉疾病,包括外周神经病,运动神经元病和肌无力综合征的小鼠模型NMJ活动。

关键字:神经肌肉接头, 电生理, 诱发终板电位(eEPP), 微型终板电位(mEPP), 神经毒素, 再生

材料和试剂

  1. 银线(世界精密仪器公司,产品目录号:AGW2030)
  2. 1ml注射器(CHEMIL s.r.l.,Padova,目录号:S01G25)
  3. 培养皿35毫米(任何生产者都很好)
  4. 用Sylgard(Dow Corning,Sylgard 184 Silicone Elastomer试剂盒)涂覆的培养皿(35×10mm)
  5. 柔性针电极Microfil(世界精密仪器公司,产品目录号:MF34G-5)
  6. 2μl微量吸管的技巧
  7. 200μl微量吸管的技巧
  8. 1,000μl微量吸管的技巧
  9. 用于细胞内微电极的玻璃毛细管(长度100mm,内径0.86mm,外径1.50mm; Science Products,目录号:GB150F-10)
  10. 用于刺激微电极的玻璃毛细管(长度100mm,内径1.05mm,外径1.50mm; Science Products,目录号:GB150TF-10)
  11. 希望的应变和年龄的小鼠
    注:我们在此使用plp-GFP C57BL / 6J转基因小鼠。
  12. 氯化铁(III)(FeCl 3)(Sigma-Aldrich,目录号:451649)
  13. 氯化银(AgCl)(Sigma-Aldrich,目录号:204382)
  14. μ-芋螺毒素GIIIB(Alomone,耶路撒冷,以色列)
  15. 碳酸氢钠(NaHCO 3)(Sigma-Aldrich,目录号:S5761)
  16. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
  17. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5655)
  18. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S3014)
  19. 氯化镁,标准溶液1M(MgCl 2)(Honeywell International,Fluka,目录号:63020)
  20. 氯化钙二水合物(CaCl 2·2H 2 O)(Sigma-Aldrich,目录号:C5080)
  21. 氯化氢(HCl)(Sigma-Aldrich,目录号:H1758)
  22. 乙酸钾(CH 3 COOK)(Sigma-Aldrich,目录号:P3542)
  23. 林格的解决方案(见食谱)
  24. 记录电极溶液(见食谱)

设备

  1. 微量移液器
  2. 容量瓶(通常500毫升;任何生产者都可以)
  3. 电生理学设置完成与防震台(纽波特,美国)(图3)
  4. 用于电生理学设置的立体显微镜(Leica Microsystem,型号:Leica MZ125,数值孔径0.8,计划消色差物镜1.6x)(图3中#1)
  5. 用于细胞内记录电极的液压显微操作器(NARISHIGE,型号:MHW-103,三轴水力显微操作器)(图3中的#2)
  6. 刺激电极显微操作器(手动显微操作器,MärZHÄUSERWETZLAR,型号:MM 33)(图3中#3)
  7. 法拉第笼(图3中#4,自制)和刺激器:S88刺激器(Grass,Warwick,RI,USA)(图3中#5)
  8. 放大器:细胞内桥式放大器(Npi electronic,型号:BA-01X)(图3中#6)
  9. 2镊子(微珠宝镊子,鲁道夫医学,目录号:RU 4240-05)
  10. 剪刀(微弹簧剪刀,Rudolf Medical,VANNAS,目录号:RU 2260-08)
  11. 剪刀(精致手术剪刀,Rudolf Medical,目录号:RU 1503-12)
  12. 解剖显微镜(OPTIKA显微镜,型号:SZM-LED2)
  13. 移液器拔管器(P-97火焰/布朗微管拔管器)(Sutter Instruments,型号:P-97)
  14. A / D接口(National Instruments,型号:PCI-6221)和与该软件兼容的计算机(图3中的#7)
  15. 气罐95%O 2与5%CO 2(任何尺寸和任何供应商都很好)
  16. 气缸压力调节器(Air Liquid,型号:HBS 240-1-2)

软件

  1. 录音:WinEDR免费软件(斯特拉斯克莱德大学,格拉斯哥,苏格兰,英国)
  2. 分析:Clampfit(Molecular Devices,Sunny-vale,CA,USA)

程序

  1. 解决方案和设置准备
    1. 使用移液管拉拔器从丝状硼硅酸盐玻璃(外径:1.5mm;内径0.86mm)和用于丝状硼硅酸盐玻璃(外径:1.5mm;内径1.05mm)的用于神经刺激的微电极拉出用于细胞内记录的微电极。许多电极可以拉出并储存在室温下直到使用。
      注意:拉细胞内或刺激微电极的准确设置可能因玻璃(硼硅酸盐和硅铝酸盐),玻璃毛细管壁的厚度,玻璃毛细管制造商和批次,拉片加热片尺寸,材料和时间,拉伸速度,时间,步骤,每个步骤的热量以及环境条件(湿度)。这些参数与细胞内微电极特别相关,必须准确设置,直到电极电阻达到最佳(见下文)。这需要在加热条件下仔细了解玻璃微电极的行为,并且在设置调整到新值时清楚了解拉拔器功能和响应。建议用不同的牵引器设置牵拉多个毛细血管,直到确定正确的参数。电阻可以通过利用特定的胞内放大器电路来测量,该电路根据欧姆定律显示测量的电阻。一个理想的电阻应该在10到30MΩ之间,这个值基本上对应于非常小的尖端,直径为0.5微米(见图2)。刺激电极的尖端尺寸并不重要,因为它可以通过朝着记录室底部的温和触碰将其断开来调节至神经直径(见图2A)。
    2. 通过用95%O 2,5%CO 2鼓泡至少15分钟将林格溶液(配方1)缓冲至pH 7.4。
      注意:一次标准实验需要约10 ml的溶液。
    3. 准备氯化银线。 AgCl的形成通过在10mM FeCl 3 / HCl溶液中化学氧化Ag涂覆的电极来实现。将电极浸入溶液10秒。
      注意:由于反复插入玻璃微电极会刮擦AgCl层,因此应定期重复此步骤(大约每2-3周)。
    4. 用记录电极溶液回填记录电极(配方2)。该步骤以两个连续的步骤进行:
      1. 通过加热(中等长度)用点火器预热电极几秒钟,并快速将底部浸入溶液中。使预填充电极保持水平,以使溶液通过毛细管现象向上流至尖端(图2B)。这大约需要30分钟;
      2. 使用1毫升注射器和电极Microfil毛细管,添加记录电极溶液,直到电极几乎被填充(见图2C)。
        注意:避免在电极填充过程中注入气泡。


        图2.刺激和记录电极A.刺激电极。预填充(B)和填充(C)记录电极。箭头表示解决方案填充的级别。

    5. 打开电生理设备的组件(图3)。


      图3.电生理设置组件。 1)立体显微镜专用于电生理学建立; 2)细胞内电极显微操作器; 3)刺激电极显微操作器; 4)法拉第笼; 5)刺激器; 6)放大器;和7)带有WinEDR免费软件的电脑。

    6. 连接录音(填充)和刺激电极到他们的电极持有人。在显微镜下放置装满林格溶液的培养皿,并将记录电极尖浸入溶液中。打开放大器并测试电阻。最佳值在10到30MΩ之间。
      备注:
      1. 电极电阻与尖端直径成正比(尖端越大,电阻越低)。这种关系可能会由于实验问题而改变:i)由于盐微晶体的形成或微气泡的存在,尖端的不完全填充或阻塞倾向于增加电阻。要恢复合适的值,将电极尖端浸入林格溶液中几分钟:盐溶解并且电阻降低。如果不是,轻轻地揉搓电极室底部的电极尖端,以对尖端造成'受控制的损伤'。阻力应该下降。
      2. 微电极电阻的突然增加通常是由于组织碎片(结缔组织或脂肪组织)引起的尖端阻塞。在这种情况下,可以通过传递大量正电流或负电流来清洁吸头(许多放大器具有“电极吸尘器功能”)。或者,将尖端与腔室底部摩擦也可能有所帮助。如果电阻不降低和/或记录迹线有噪音,则必须更换电极。当电阻低于10MΩ或尖端太宽(已损坏)时,电极也必须更换。
    7. 打开刺激器并设置刺激参数:对于eEPP,0.4毫秒持续时间和0.5 Hz频率的刺激是适合的。
      注意:这些参数必须进行优化以测量NMJ功能,原因有两个:a)脉冲步长持续时间最短以防止过度的刺激伪影; b)脉冲步频非常低,以防NMJ“疲劳”。
    8. 运行WinEDR免费软件。

  2. 解剖比目鱼肌
    1. 根据你的动物处理协议安乐死鼠标。
      注意:所有程序均按照意大利法律和政策(DL No. 26,2014年3月14日)执行,欧盟委员会指令2010/63 / UE并经帕多瓦大学(OPBA-Organismo Preposto al Benessere degli Animali)的兽医服务批准(第359/2015号议定书)。所有的程序应该根据进行实验的机构的道德标准来使用。
    2. 用剪刀在脚踝周围切割皮肤。
      在大腿上切下鼠标腹部(图4A)。
    3. 借助镊子,皮肤后肢肌肉。把鼠标放回仰卧位,并用针脚挡住爪子。使用小剪刀,切开跟腱(图4B)。
    4. 轻轻地拉动肌腱,用Vannas剪刀,通过切除比目鱼肌外侧的结缔组织(图4C),伴随肌肉从腓骨脱离。
      注意:
      1. mEPP和eEPP的电生理学记录可以在任何可收集的肌肉中实际上完成,而不损伤肌纤维并且其神经正确附着。在这里,我们提供比目鱼肌肉的详细程序,在我们看来,这是最容易收获的比目鱼肌肉。
      2. 将结缔组织从内侧切到外侧以避免(比目鱼)肌肉受损。


      图4.比目鱼肌收集的初始步骤。 :一种。切割皮肤在脚踝和大腿周围; B.切开跟腱; C.箭头显示切割方向以分离结缔组织避免肌肉损伤。

    5. 当整个比目鱼肌肉可见时,使用小Vannas剪刀仔细切断第二根肌腱尽可能靠近膝盖(图5A)。此时,比目鱼肌对跟腱退缩,肌肉附着的肌肉都可以切除而不影响比目鱼肌的完整性(图5B)。
    6. 将肌肉置于Sylgard底部的培养皿中,并充满氧化林格溶液。如图5C所示将它们固定,使比目鱼肌朝向操作员(黑色箭头)。将培养皿置于立体显微镜下进行下一步操作。
      注意:对于解剖,Ca 2 +游离Ringer's溶液对于防止由于机械刺激引起的肌肉收缩或挛缩是可取的。


      图5.收集下肢后肢肌肉箭头表示比目鱼肌。

    7. 使用镊子,通过抓住下部肌腱轻轻抬起比目鱼肌,并切割连接组织使其与腓肠肌连接。继续直至神经可见(图6A)。神经位于比目鱼的下面,看起来像插入肌纤维的白线(图6A-6C中的虚线)。
    8. 使用Vannas剪刀清理结缔组织的神经(图6B)。
      注意:最佳刺激需要大约0.5厘米的神经长度。
    9. 当比目鱼肌及其神经完全自由时,切开跟腱水平的腓肠肌(图6C)。


      图6.比目鱼的神经分离和解剖仍然是相关的。虚线表示神经; S是比目鱼肌,G是腓肠肌。

    10. 通过两根肌腱将比目鱼肌固定在培养皿底部(图7A)。
      注意:必须固定比目鱼肌以拉伸肌肉并促进记录电极进入肌纤维。避免过度拉伸,这可能会导致肌肉纤维受损。


      图7.实验室的细节A.肌肉定位到培养皿中; B. 1)记录电极; 2)刺激电极; 3)地面; 4)充氧管; 5)参比电极; 6)比目鱼肌。

    11. 轻轻地,从残余组织碎片中清除肌肉和神经,而不会损伤纤维或神经。
    12. 用林格溶液冲洗肌肉一至两次,以消除结缔组织碎屑或皮毛。将肌肉留在装满2毫升新鲜林格溶液的培养皿中。

  3. 神经刺激和EPP记录
    1. 将培养皿放置在电生理学设备的立体显微镜下,并浸没连接到O 2 / CO 2气罐的管以使氧气在溶液中起泡(图7B)。
    2. 将地面和刺激电极的参考放在溶液中(图7B)。注意:需要接地以消除由于法拉第笼内部设备之间的电流回路而产生的电气噪声(法拉第笼需要防止电网电流噪声)。需要参考来测量肌纤维膜两端的电压,即静息电位以及它如何变化(mEPP,eEPP和动作电位)。通过比较记录电极的电压和参考物的电压来测量膜电位,所述参考物必须与肌肉周围的细胞外液(溶液)接触。
    3. 使用显微操作器,将刺激电极置于溶液中并吸取林格溶液(填充大约一半的电极体积)。然后,将电极靠近神经残端并将其与溶液一起吸入电极(图8)。
      注意:使用注射器产生负压以抽吸溶液并吸入神经。必须将神经完全浸入溶液中以避免空气进入电极并提供电极与神经之间的电连续性。

      “”src
      图8.电极的定位A.记录电极1)比目鱼肌2)记录电极3)神经。 B.刺激电极。 1)比目鱼肌,2)记录电极,3)神经和4)记录电极。

    4. 通过增加电压强度刺激神经,直到肌肉收缩。对于细胞内记录,将刺激器设置为实现肌肉收缩所需值的1.5倍的电压强度。
      注意:肌肉收缩通常是在5到10V之间的电压下实现的。如果15V内没有达到收缩,神经可以被推出并再次吸入电极。或者,更换销钉以减少肌肉拉伸。如果收缩仍不可见,可能是在解剖过程中神经受伤。避免电压高于20 V,因为肌肉收缩可能是由肌肉直接刺激触发的。
    5. 通过使用细胞内电极显微操作器,将记录电极的尖端放入腔室的林格溶液中并打开放大器。将电压偏移到0.将电极尖端移动到图9所示盒装区域内的肌肉纤维附近,其中神经进入肌肉并且NMJ集中。当电极的尖端接近肌纤维时,观察到电压的轻微极化(约-20mV)。
      注意:尽管不是绝对必要的,但运用荧光报告基因的转基因小鼠在运动神经元或Schwann细胞中偶联荧光立体显微镜有助于更准确地发现NMJ。


      图9. NMJ的分布启动子(plp-GFP(Mallon等人,2002))下在Schwann细胞中表达GFP的C57 / BL6 / J转基因小鼠的比目鱼肌,用落射荧光立体显微镜成像。使用荧光施万细胞,NMJ可以很容易地在神经轴突末端(白色圆圈)被发现。

    6. 用显微操作器将记录电极向肌肉深处移动。当肌纤维正确穿刺时,从放大器或示波器/计算机接口读取的电压突然下降到非常负的值,对应于肌纤维的静息电位(V rest)一般在-90到-60mV之间)。
      注意:
      1. V Rest 的值因细胞而异,在某些情况下则因治疗而异。在这种情况下,记录来自骨骼肌细胞,其中V rest在-60和-90 mV之间。为了获得可比较的eEPP幅度(eEPP幅度线性地依赖于V rest(Boyd和Martin,1955)),V rest被任意设定为-70mV以通过使用电流来标准化注射命令。
      2. V Rest 是NEGATIVE。正电位可能对应于光纤外部电极的错位。
      3. 如果静息电位在-20和-40 mV之间,电极没有正确插入肌肉细胞。放大器通常配备'BUZZ功能',这是一种基于过补偿电容补偿系统引起的振荡的电路,有助于电极进入光纤。
      4. 如果膜电位逐渐去极化(即,缓慢返回到0),肌纤维膜可能被损坏并泄漏或电极脱落。改变纤维。
      5. 在任何情况下,损坏的纤维都不会显示预期的膜电位,从而改变纤维。
      6. 延长去神经支配,如用肉毒杆菌神经毒素或神经切断治疗的情况下,增加静息膜电位。常见的结果是肌纤维颤动,肌纤维的自发性,随机性和异步性收缩(Purves和Sakmann,1974; Moravec和Vyskocil,2005)。原纤维化可被特定药物抑制(见下文)。
    7. 此时,可以记录mEPP。
      1 :在刺激时,动作电位被激发并且可以被记录。相反,eEPP不能被记录,因为它们被动作电位隐藏/覆盖。
      2 :请考虑动作电位会导致肌肉收缩,这可能会破坏记录电极或导致其从光纤中退出录制。
    8. 添加μ-芋螺毒素GIIIB以选择性地抑制负责触发突触后动作电位的电压门控Na + +通道(Nav1.4主要由肌肉表达),从而停止肌肉收缩。使用微量移液器,向培养皿中加入1μlμ-芋螺毒素原液(1 mM),并开启氧合。
    9. 15分钟后,刺激并检查肌肉收缩。如果肌肉没有收缩,可以记录EPP,否则再等5-10分钟。
      注意:更深的肌肉纤维可能需要更多时间用于μ-芋螺毒素活性。
    10. 停止氧合作用(由于起泡或过度流动引起的溶液运动将人为因素引入记录)并开始刺激。
      注意:eEPP幅度与神经递质释放量成正比,而释放量又与刺激后释放的突触囊泡的数量成正比。因此可以使用eEPP来测试神经末梢的功能状态,从而提供对突触前效应的准确估计。
    11. 设置记录时间(至少30秒)并记录电流钳模式下的eEPP。对每个肌肉打十几根纤维重复该过程(如果eEPP在同一肌肉内是异质的,则增加纤维采样)。典型的记录轨迹如图10所示,其中刺激伪影(与刺激幅度成比例),EPP与神经末端活性和mEPPs成正比。


      图10.采集期间的典型痕迹

数据分析

  1. WinEDR软件提供EDR文件格式。使用WinEDR软件,将该文件导出为与Clampfit软件兼容的ABF文件格式。
    注意:数据分析的步骤可能因用于记录和分析的软件而异。
  2. 用Clampfit软件打开ABF文件并在控制轨迹上建立一个模板。在控制轨迹中定义eEPP的形状将'教导'Clampfit软件在所有其他文件中自动识别eEPP(或mEPP)的形状。分析所有要与同一模板进行比较的分析文件。 Clampfit软件为跟踪中的每个事件提供一个表格,其中定义了EPP格式的参数(开始时间,结束时间,峰值幅度,面积等等。 )。
    注:EPP的一般形状是抛物线。初始斜率与发射器的突触后响应的平均幅度有关(突触后效应);曲率的程度与递质释放的可能性(突触前功效)有关;该幅度与突触前终端中独立突触前释放位点的数目有关。通常,eEPP和mEPP幅度之间的比率被计算为eEPP的“量子含量”,即在突触前动作电位之后发生融合的囊泡的数量。理论上,不同实验条件下的不同曲线的视觉比较提供了关于哪些突触参数被改变的见解。

食谱

  1. 林格的解决方案

    注意:
    1. 使用ddH 2 O。所有使用的玻璃器皿必须用0.1M氯化氢洗涤以除去任何痕量的碳酸盐,并冲洗得很好。一旦准备好,这种解决方案可以储存在4°C,但不超过3个月。如果存在沉淀物或乳光,则丢弃并制备新的溶液。
    2. 在分析之前,林格溶液用95%O 2,5%CO 2,5%通过曝气至少15分钟以获得7.4的pH水平。
  2. 记录电极解决方案

致谢

这项工作得到了帕多瓦大学的支持,授予M. Pirazzini授予“未在帕多瓦大学就业的年轻人的高级研究资助”,并向S. Negro授予“初级奖学金”,Fondazione Caritro授予'Bando 2017 giovani ricercatori coinvolti in progetti di eccellenza'授予G. Zanetti。所有程序均在由生物医学科学系(帕多瓦大学)Cesare Montecucco教授领导的'神经毒素,神经麻痹和再生'实验室中进行。作者声明没有竞争利益。 SN和GZ在AM的帮助下进行比目鱼肌肉制备的程序。 SN和GZ拍照描述所有的程序并准备好数字。 GZ和MP在SN的帮助下写了这篇论文。所有作者都回顾了手稿并批准了最终版本。

参考

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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zanetti, G., Negro, S., Megighian, A. and Pirazzini, M. (2018). Electrophysiological Recordings of Evoked End-Plate Potential on Murine Neuro-muscular Synapse Preparations. Bio-protocol 8(8): e2803. DOI: 10.21769/BioProtoc.2803.
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