Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

Podsumowanie

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

Streszczenie

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Wprowadzenie

The retinotectal circuit is the major component of the amphibian visual system. It is comprised of the RGCs in the eye, which project their axons to the optic tectum where they form synaptic connections with postsynaptic tectal neurons. The Xenopus tadpole retinotectal circuit is a popular developmental model to study neural circuit formation and function. There are many attributes of this tadpole's retinotectal circuit that render it a powerful experimental model^1,2,3. One major attribute, and the focus of this article, is the ability to carry out whole cell patch clamp recordings from tectal neurons, in vivo or using a whole brain preparation. With an electrophysiology rig outfitted with an amplifier that supports voltage- and current-clamp recording modes, whole cell patch clamp recordings allow a neuron's electrophysiology to be characterized at high resolution. As a result, whole cell patch clamp recordings from tectal neurons across the key stages of retinotectal circuit formation have provided a detailed and comprehensive understanding of the development and plasticity of intrinsic^4,5,6,7 and synaptic^8,9,10,11 properties. Combining whole cell patch clamp tectal neuron recordings, the ability to express genes or morpholinos of interest in these neurons¹², and a method to assess visual guided behavior via an established visual avoidance test¹³ promotes the identification of links between molecules, circuit function, and behavior.

It is important to note that the type of high resolution data acquired from whole cell patch clamp recordings is not possible using newer imaging approaches such as the genetic calcium indicator GCaMP6, because although using calcium indicators permits the imaging of calcium dynamics across large populations of neurons simultaneously, there is no direct or obvious way that the specific electrical parameters can be obtained by measuring delta fluorescence in the somata, and there is no way to voltage clamp the neuron to measure current-voltage relationships. Clearly these two distinct approaches, electrophysiological recordings and calcium imaging, possess non-overlapping strengths and generate different types of data. Thus, the best approach depends on the specific experimental question being addressed.

Here, we describe our method for acquiring whole cell patch clamp recordings from neurons of the tadpole optic tectum using an in vivo preparation, whole brain preparation, and a newer modified whole brain preparation that was developed in our lab¹⁴. In the Representative Results section, we demonstrate the experimental advantages of each preparation and the different types of data that can be obtained. The limits and strengths of the different preparations, as well as tips for troubleshooting, are included in the Discussion section.

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Protokół

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Wyoming. All procedures, including electrophysiological recordings, are carried out at room temperature, approximately 23 °C. All methods described here are optimized for recording tectal neurons from tadpoles between developmental stage 42 and 49 (staged according to Neiuwkoop and Faber¹⁵).

1. In Vivo Preparation

1. Anesthetize the tadpole.
   1. Place the tadpole in a small Petri dish containing Steinberg's solution with 0.01% MS-222 for approximately 5 min.
      ​NOTE: MS-222 aka "Tricaine" is a common fish and amphibian anesthetic. The tadpoles are raised in the Steinberg's solution in mM: 0.067 KCl, 0.034 Ca(NO₃)₂•4H₂O, 0.083 MgSO₄•7H₂O, 5.8 NaCl, 4.9 HEPES.
   2. Ensure the tadpole is deeply anaesthetized (non-responsive and no longer swimming) before proceeding to step 2.
2. Secure the anesthetized tadpole to a submerged silicone block on the floor of the dissecting/recording dish.
   1. Use a disposable transfer pipette to move the anaesthetized tadpole to a dissection/recording dish containing external recording solution (115 mM of NaCl, 2 mM of KCl, 3 mM of CaCl₂, 3 mM of MgCl₂, 5 mM of HEPES, 1 mM of glucose; adjust pH to 7.25 using 10 N of NaOH, osmolarity 255 mOsm).
      1. To minimize spontaneous muscle twitches, add the acetylcholine receptor blocker, tubocurarine (100 µM) to the external solution. (Typically, this is done by using a 200 µL pipette to add 100 µL of a 10 mM tubocurarine stock to 10 mL of external solution).
   2. Using insect pins, secure the tadpole, dorsal side up, to a submerged block of silicone elastomer (such as Sylgard 184, see Materials Table for details), which has been glued to the dissecting dish floor.
      Note: The placement of the pins is critical: place them on either side of the brain, sufficiently caudal to avoid impaling the afferent RGC axons that project from the eye and enter the brain just anterior to the optic tectum (Figure 1A).
3. Fillet the brain along the midline.
   1. For a clear view of the brain, remove the skin overlying the brain by making a superficial incision along the midline using a sterile 25-G needle.
   2. Fillet the brain along the same midline axis by inserting the needle into the neural tube and pulling gently upward (dorsally) such that the dorsal portion of the tube is cleanly cut (severed) while leaving the floor plate intact (Figure 1B).
      ​NOTE: It is important that the floor plate of the neural tube be left intact because the afferent sensory inputs enter the tectum via the floor plate.
4. Remove the transparent ventricular membrane that covers the tectal neurons.
   1. Move the recording dish to the electrophysiology rig and use a broken glass pipette tip to remove the ventricular membrane overlying the tectal neuron cell bodies. Break the tip by lightly dragging it across a delicate task wiper.
   2. Screw the pipette into the pipette holder and lower it down to the optic tectum via the micromanipulator.
   3. Use the broken pipette to peel the ventricular membrane away from the tectum.
      NOTE: It is not necessary to remove the membrane from the entire tectum; a small window will suffice and provide access to plenty of neurons.
5. Obtain the whole cell patch clamp recordings.
   NOTE: The approach from this point forward is similar to carrying out whole cell patch clamp recordings from mouse brain slices as described in Segev, et al.¹⁶(Figure 1C).
6. Record the tectal neuron responses to a whole field flash of light projected onto the retina.
   1. To project a whole field flash of light onto the retina, place an optic fiber adjacent to the tadpole's eye. At the other end of the optic fiber is an LED in line with a variable resistor that allows for the light luminance to be controlled. Trigger the LED by the digital output of the amplifier. In this way, whole field flashes of varying intensities of light can be recorded (Figure 4A).

2. Whole Brain Preparation

1. Perform steps 1.1 to 1.3.
   Note: There is no need for tubocurarine in the external solution for this preparation.
2. Isolate the brain.
   1. Use a 25-G needle to sever the hindbrain (Figure 2A).
   2. To isolate the whole brain from the tadpole, gently run the needle underneath the brain, in a caudal-to-rostral direction to sever all lateral and ventral connective tissue and nerve fibers.
3. Secure the brain to a block of silicone elastomer.
   1. Once completely freed, secure the brain to a block of silicon elastomer by placing one pin through one of the olfactory bulbs and another pin through the hindbrain (Figure 2B). This is the optimal configuration for recording from tectal neurons.
4. Move the dish containing the pinned whole brain preparation from the dissecting scope to the electrophysiology rig. Remove the ventricular membrane using a broken glass pipette as described in step 1.4.
5. Place a bipolar stimulating electrode on the optic chiasm (where the axon tracts from each eye cross at the midbrain) to directly activate the RGC axons.
   1. Place the bipolar stimulating electrode rostral, and almost adjacent to, the large middle ventricle. The optic chiasm is located immediately rostral to the large middle ventricle (Figure 2B).
      1. Gently lower the stimulating electrode down onto the optic chiasm such that a small dent is formed in the tissue. The bipolar electrode is driven by a pulse stimulator which allows the strength of stimulation to be precisely controlled.
6. Perform the whole cell patch clamp recording (Figure 2C) as described by Segev, et al.¹⁶

3. Horizontal Brain Slice Preparation

1. Perform steps 2.1 to 2.3.
2. Use a razor blade to excise the most lateral fourth (which in vivo corresponds to the most dorsal fourth) of one side of one optic tectum. This cut is made parallel to the rostral-caudal plane as shown in Figure 3A.
3. Re-pin the brain to the side of the silicone elastomer with the sliced side facing up (so that the somata and neuropil can be directly accessed for recording) and the ventricular surface of the brain facing away from the silicone elastomer block (Figure 3B) (so that a bipolar electrode can be placed on the optic chiasm).
4. Perform the whole cell patch clamp or local filed potential recording (Figure 3C) as described by Segev, et al.¹⁶

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Wyniki

To record light-evoked responses a whole field flash of light is projected onto the retina while the resulting response is recorded from individual tectal neurons (Figure 4A). This particular protocol is designed to measure both the response of the neuron to the light turning on ("On" response) and then turning off 15 s later to measure the "Off response." Tectal neurons typically exhibit robust On and Off responses (show...

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Dyskusje

All methods described in this work are optimized for recording tectal neurons from tadpoles between developmental stage 42 and 49 (staged according to Neiuwkoop and Faber¹⁵). By stage 42, the tadpoles are sufficiently large and sufficiently developed so that the insect pins can be placed on either side of brain for in vivo recordings and for carrying out the whole brain dissection. At earlier stages, when the tadpoles are essentially two-dimensional (i.e., flat), the approaches d...

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Materiały

Name	Company	Catalog Number	Comments
Stemi Stereo 508	Zeiss	495009-0006-000	Dissecting microscope
MS-222 "Tricane"	Finquel	ARF5G	Amphibian general anesthetic
Sodium Chloride (NaCl)	Fisher Scientific	S271-3	Used to prepare Stienberg's solution and external solution
Potassium Chloride (KCl)	Fisher Scientific	P217-500	Used to prepare Stienberg's solution and external solution
HEPES	Sigma-Aldrich	H3375-1KG	Used to prepare Stienberg's solution and external solution
Calcium nitrate tetrahyrate (Ca(NO3)•4H2O)	Sigma-Aldrich	237124-500G	Used to prepare Stienberg's solution
Magnesium Sulfate (MgSO4)	Mallinckrodt Chemicals	6066-04	Used to prepare Steinberg's solution
Calcium Chloride (CaCl2)	Sigma-Aldrich	C5080-500G	Used to prepare external recording solution
Magnesium Chloride (MgCl2)	J.T. Baker	2444-01	Used to prepare external recording solution
D-glucose Anhydrous	Mallinckrodt Chemicals	6066-04	Used to prepare external recording solution
Tubocurarine hydrochloride pentahydrate	Sigma	T2379	Nicotinic acetylcholine receptor antagonist
Insect Pins	Fine Science Tools	26002-10	0.1mm diameter stainless steel pins
Sylgard 184 Silicone Elastomer Kit	Dow Corning	761028	Preweighed monomer and curing agent kit
Sterile Polystyrene Petri Dish - 60x15mm	Fisher Scientific	AS4052	Small petri dishes
PrecisionGlide Needle 25Gx5/8 (.0.5mm X 16mm)	BD	305122	Syringe needles
1mL Slip Tip Tuberculin Syringe	BD	309659	Disposable, sterile syringes
Borosilicate pipette glass	Sutter Instrument	BF150-86-10HP	Pulled to desired specifications using pipette pulling machine
Flaming/Brown Micropipette Puller	Sutter Instruments	P-97	Fabricates micropipettes for electrophysiology recording
Kimwipes Kimtech wipes	Kimberly-Clark	34120	Delicate task lint-free wipers
Axon Instruments MultiClamp 700B Headstage CV-7B	Molecular Devices	1-CV-7B	Current clamp and voltage clamp headstage
MP-285 Motorized Manipulator with Tabletop Controller	Sutter Instrument	MP-285/T	Control for headstage on electrophysiology rig
Fiber-Coupled LED (Green)	Thorlabs	M530F2	Fiber optic cable paired with green LED
Cluster Bipolar Electrode (25µm diameter)	FHC	30207	Bipolar stimulating electrode
ISO-Flex Stimulator	A.M.P.I. (Israel)	Contact manufacturer	Flexible stimulus isolator
Axon Instruments 700B Multipatch Amplifier	Molecular Devices	2500-0157	Amplifier for voltage- and current-clamp recording
Digidata 1322A digitizer	Molecular Devices	2500-135	Data acquisition system for electrophysiology recording
Axio Examiner.A1	Zeiss	491404-0001-000	Microscope for electrophysiology
Micro-g Lab Table	TMC	63-533	Air table for electrophysiology microscope
Inspiron 620 Personal Desktop Computer with Windows 7 64-bit	Dell	D06D001	Computer running electrophysiology software
c2400 CCD camera	Hamamatsu	70826-5	Charge-coupled device camera for electrophysiology imaging
7 O'Clock Super Platinum Stainless Razorblades	Gillette	CMM01049	Platinum-coated stainless razor blades
Transfer Pipets	Fisher Scientific	13-711-7M	Disposable Polyethylene transfer pipets