Materials and Reagents

Note: The materials and equipment listed refer to the equipment used in Städele and Stein (2016). The principles of retrograde extracellular axon stimulations are universal and the procedures can be easily adapted to other preparations. To reduce costs, comparable materials, equipment and software may be used that serve the same functions. For the general public or a teaching classroom, we suggest utilizing equipment from Backyard Brains (https://backyardbrains.com).

1. Syringe, filled with petroleum jelly for preparing extracellular recording and stimulation wells
   For preparing syringes, the following materials will be needed:
   1. Petroleum jelly (100% pure, pharmacy)
      
   2. 100 ml glass beaker (for melting petroleum jelly)
      
   3. 5 ml Luer lock syringe (e.g., BD, catalog number: 309603 )
      
   4. Injection needle (20 G x 1.5”, e.g., Santa Cruz Biotechnology, catalog number: sc-359535 )
      
   5. Sand paper (80 to 100 grit, hardware store)
      
2. Recording/stimulation electrodes
   For preparing electrodes, the following materials will be needed:
   1. Stainless steel wire, uncoated (A-M Systems, catalog number: 794800 )
      Low-cost alternative: Minutien pins (see below) or sewing pins
      
   2. Electrical wire, red and black PVC insulated (Southwire, model: 22 gauge stranded, catalog number: 57572444 , hardware store)
      
   3. Wire stripper (hardware store)
      
   4. Needle-nose pliers (hardware store)
      
   5. Heat shrink tubing (hardware store)
      
   6. Tin solder, 3/32 in. (Forney, catalog number: 38109 )
      
3. Petri dish lined with silicon elastomer (e.g., Sylgard 184, Sigma-Aldrich, catalog number: 761036 ; or Elastosil RT 601, Wacker Chemie, catalog number: 60063613 )
   
4. Minutien pins (Fine Science Tools, catalog number: 26002-10 )
   
5. Modeling clay (craft store)
   
6. Dissected nervous system
   Note: We are using adult Jonah crabs (Cancer borealis), purchased from The Fresh Lobster Company (Gloucester, MA).
   
7. Physiological saline (see Recipes)
   The recipe for C. borealis saline can be found in Table 1
   1. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9625 )
      
   2. Magnesium chloride hexahydrate (MgCl₂·6H₂O) (Sigma-Aldrich, catalog number: M9272 )
      
   3. Calcium chloride dihydrate (CaCl₂·2H₂O) (Sigma-Aldrich, catalog number: C7902 )
      
   4. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
      
   5. Trizma base (Sigma-Aldrich, catalog number: T1503 )
      
   6. Maleic acid (Sigma-Aldrich, catalog number: M0375 )
      

Equipment

1. Heating plate (Thermo Fisher Scientific, Thermo Scientific^TM, model: Nuova^TM Stirring HotPlates, catalog number: SP18425Q )
   
2. Stereomicroscope (e.g., Leica Microsystems, model: MS5 )
   
3. Stimulator (A.M.P.I, model: Master8 Pulse Stimulator )
   Low cost alternative: Pulse Pal V2 (Sanworks, catalog number: 1102 )
   
4. Amplifier (A-M Systems, model: Differential AC Amplifier 1700 , catalog number: 690000)
   Low cost alternative: Spikerbox (Backyard Brains, model: Neuron SpikerBox )
   
5. Data acquisition board (CED, model: Power 1401-3A )
   Low-cost alternative: by using the BYB Spike Recorder, data can be digitized by using the microphone jack and soundcard on a computer/laptop. A second low-cost alternative is Spikehound (http://spikehound.sourceforge.net), which also allows recording through the computer soundcard
   
6. Soldering station (Apex Tool, Weller, model: Station 50/60W 120 V WES51 , catalog number: WES51)
   
7. Voltmeter (FLIR Systems, Extech, model: EX330 , catalog number: 203489911)
   
8. Forceps (e.g., Fine Science Tools, model: Dumont #5, catalog number: 11251-10 )
   

Software

1. Recording software (Spike2 version 7.12, Cambridge Electronic Design Limited)
   Low cost alternative: BYB Spike Recorder (freeware, available on https://backyardbrains.com/products/spikerecorder) or Spikehound (http://spikehound.sourceforge.net)
   

Procedure