Implanting an Electrode Array and Optic Fiber into the Mouse Hippocampus

Abstract

Source: Bender, F., et. al., Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice. J. Vis. Exp. (2018)

This video demonstrates the stereotactic implantation of a wire electrode array and an optic fiber in the hippocampus of a mouse model.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of Optic Fibers (Figure 1A)

1. Use multimode optic fiber (105 µm core, glass-clad with silica core, 0.22 NA). Strip the 125 µm cladding of the fiber core using a micro-stripper while the fiber is still attached to the fiber spool.
2. Cut the fiber to a length of approximately 2-3 cm using a diamond knife.
3. Insert the fiber in a zirconia ceramic stick ferrule (ID: 126 µm). Approximately 0.5-1 mm of the optic fiber should protrude from the convex side of the ferrule.
4. Using a needle, apply one drop of epoxy glue to both ends of the ferrule but not onto the sides of the ferrule. Alternatively, use super glue.
5. Allow the glue to dry for at least 30 min.
6. Polish the convex side of the ferrule using diamond lapping fiber polishing film (3 µm grits).
7. Test the fidelity of light transfer using an optical power meter.
   1. Set the wavelength on the power meter to the same wavelength as the laser being used.
   2. Position the patch cord with the tip facing the center of the sensor. Power on the laser and read the light output from the power meter. Record the value.
   3. Connect the optic fiber to the patch cord via a mating sleeve and position it with the tip of the fiber facing the center of the sensor. Power on the laser and read the light output from the power meter. Record the value.
   4. Calculate the transmission rate by dividing the second value by the first value. If the transmission rate is below 0.5, discard the fiber; otherwise, use it for implantation.
   5. Test the transmission rate for each fiber before implantation.
   6. For later experiments, adjust the laser's light intensity output to the transmission rate of the optic fiber: Set the light output from the patch cord tip to 5-15 divided by the transmission rate to achieve a final light output from the fiber tip of 5-15 mW.

2. Preparation of Tungsten Wire Arrays for LFP Recordings (Figure 1B)

1. Glue several (for example 6) 100 µm silica tube guides in parallel to the sticky side of a piece of tape. Cut one piece, approximately 4-6 mm, for one wire array assembly.
2. Thread formvar-insulated 45 µm tungsten wires through the guide tubes using forceps.
3. Strip six enamel-insulated fine copper bonding wires (approximately 5 mm long) and a grounding wire (approximately 2-3 cm long) by using a scalpel to scrape away the insulation on both ends. Solder them to the nanoconnector pins.
4. Connect each bonding wire to one tungsten wire using one drop of silver conductive paint, respectively. Let dry for at least 30 min.
5. Apply a minimum amount of cement to cover the wires. Do not apply cement to the tungsten wires, which will be inserted in the brain tissue, or to the upper part of the nano connector. Let the cement dry for at least 30 minutes.
6. Perform an angular cut (5-20°) of the tungsten wires using blunt stainless-steel scissors to enable reliable implantation of wires below or above the zone ventrally adjacent to the stratum pyramidale, where theta amplitude is too low to estimate the entrainment fidelity.
7. Deinsulate the tip (approximately 2 mm) of the ground wire by using a scalpel to scrape away the insulation. Treat it with flux and pre-solder.
8. Check potential cross-talks between electrodes using a digital multimeter. Connect the connector pins to the multimeter, which must be set to the resistance measurement mode. Check pairwise combinations of channels; a reading on the multimeter below 5 MΩ indicates significant cross-talk.
9. Use an impedance meter to check the impedance of each wire electrode in saline. Typical impedance values are below 100 kΩ.
10. To facilitate implantation, glue one optic fiber to the wire array so that the tip of the fiber is at the level of the shortest wire and the fiber tip is in close proximity but not touching the tungsten wires. Keep the angle of the fiber as small as possible in order to prevent tissue damage during implantation.

3. Stereotaxic Implantations

1. Remove the connective tissue from the top of the skull and thoroughly push down the neck muscles by approximately 2 mm to prevent muscle artifacts during the recording.
2. Clean the skull using a cotton-tip applicator and saline, and drill four holes (2 in the front and two above the cerebellum, 0.8 mm diameter) to place bone stainless-steel screws (00-96x1/16) for ground and stabilization of the implant (Figure 1D). Position the ground screw, connected to one copper wire (approximately 2-3 cm in length) above the cerebellum.
3. Cover the ground-screw completely with cement to prevent muscle artifacts during the electrophysiological recordings. Build a cement ring connecting all screws (Figure 1E).
4. Perform a craniotomy above the implantation side (Hippocampus, AP -1.94, L 1.4, V 1.4 in reference to the bregma). Apply approximately 5 µL of sterile NaCl on the surface of the brain tissue.
5. Slowly lower the wire array using stereotaxis in the craniotomy. For unitary recordings, implant a silicone probe instead of a wire array. To prevent optoelectric light artifacts, implant the optic fiber separately in the hippocampus with the fiber tip not directly facing the probe (Figure 1C—I). For investigation of the coordination of the entrainment between hemispheres, implant an additional optic fiber in the contralateral hippocampal CA1 area.
6. To protect brain tissue, apply approximately 5 µL of warm liquid wax/paraffin oil, preheated at 70 °C, with a syringe above the implantation site.
7. Apply cement around the wire array and cover the skull with cement.
8. Apply one drop of flux to the pre-soldered ground/reference wire and the pre-soldered wire connected to the ground screw using, for instance, a needle, and fuse the wires using a soldering machine.
9. Cover the entire ground wire with cement.
10. Administer 0.3 mL Erycinum (1:4 in sterile NaCl) and carprofen (5mg/mL) i.p. after surgery and for at least the two days following. The mouse typically wakes up within 15 min following surgery. Warm the animal with a red lamp to speed up recovery.
11. Monitor the mouse's weight daily for the first week following the surgery or until it is stable. Weight loss should not exceed 10% of the mouse's recorded weight before surgery. To accelerate weight stabilization, supply the mouse with wet food and condensed milk during the first days following the surgery.
12. To record hippocampal cellular activity during entrainment, implant a silicone probe in the hippocampus (AP -1.94, L 1.4, V 1, with subsequent lowering) (Figure 1C—I). Implant the optic fiber in the hippocampus at AP -3, L 1.4, V 1.6, 39° caudal-rostral. If stimulation of cell somata is desired, implant an additional optic fiber in the MS (AP +0.98, L 1, V 3.9, 15° lateral).

Results

Figure 1: Illustration of optic fibers, electrodes and surgery. (A) Illustration of an optic fiber. (B) Illustration of a wire array glued to an optic fiber for the recording of hippocampal LFP during entrainmen...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PV-Cre mice	The Jackson Laboratory	B6;129P2-Pvalbtm1(cre)Arbr/J
Stereotaxis	David Kopf Instruments, Tujunga, CA, USA	Model 963	Ultra precise small animal stereotaxic instrument
Drill bits, 0.8 mm	Bijoutil, Allschwil, Switzerland	49080HM
Soldering station	Weller Tools GmbH, Besigheim, Germany	WSD 81
Erythromycin	Rotexmedica GmbH, Trittau, Germany	PZN: 10823932	1g powder for solution for infusion
Precision drill/grinder	Proxxon, Wecker, Luxemburg	fbs 240/e
Multimode optic fiber	ThorLabs, Dachau, Germany	FG105LCA	0.22 NA, Low-OH, Ø105 µm Core, 400 - 2400 nm
Ceramic stick ferrule	Precision Fiber Products, Milpitas, CA, USA	CFLC126	Ceramic LC MM Ferrule, ID 126um
Polishing paper	Thorlabs	LF3D	6" x 6" Diamond Lapping (Polishing) Sheet
Power meter	Thorlabs	PM100D	Compact Power and Energy Meter Console, Digital 4" LCD
Multimode fiber optic coupler	Thorlabs	FCMM50-50A-FC	1x2 MM Coupler, 50:50 Split Ratio, 50 µm GI Fibers, FC/PC
Fiberoptic patch cord	Thorlabs	FG105LCA CUSTOM-MUC	custom made, 3 m long, with protective tubing, Tubing: FT030, Connector 1: FC/PC, Connector 2: 1.25mm (LC) Ceramic Ferrule
Sleeve	Precision Fiber Products, Milpitas, CA, USA	ADAL1	Ceramic Split Mating Sleeve for Ø1.25 mm (LC/PC) Ferrules
Tungsten wires	California Fine Wire Company, Grover Beach, CA, USA	CFW0010954	40 µm, 99.95%
Capillary tubing	Optronics	1068150020	ID: 100.4 µm
Omnetics nanoconnector	Omnetics Connector Corporation, Minneapolis, USA	A79038-001
Screws	Bilaney, Düsseldorf, Germany	00-96x1/16	stainless-steel
Silver conductive paint	Conrad electronics, Germany	530042
Liquid flux	Felder GMBH Löttechnik, Oberhausen, Germany	Lötöl ST	DIN EN 29454.1, 3.2.2.A (F-SW 11)