A Method for Systematic Electrochemical and Electrophysiological Evaluation of Neural Recording Electrodes

Summary

Different electrode coatings affect neural recording performance through changes to electrochemical, chemical and mechanical properties. Comparison of electrodes in vitro is relatively simple, however comparison of in vivo response is typically complicated by variations in electrode/neuron distance and between animals. This article provides a robust method to compare neural recording electrodes.

Abstract

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant characteristics. This precludes a rational selection of a particular implant as optimal for a particular application and the development of new materials based on the most critical performance parameters. This article develops a protocol for in vitro and in vivo testing of neural recording electrodes. Recommended parameters for electrochemical and electrophysiological testing are documented with the key steps and potential issues discussed. This method eliminates or reduces the impact of many systematic errors present in simpler in vivo testing paradigms, especially variations in electrode/neuron distance and between animal models. The result is a strong correlation between the critical in vitro and in vivo responses, such as impedance and signal-to-noise ratio. This protocol can easily be adapted to test other electrode materials and designs. The in vitro techniques can be expanded to any other nondestructive method to determine further important performance indicators. The principles used for the surgical approach in the auditory pathway can also be modified to other neural regions or tissue.

Introduction

Neural implants are being used increasingly for research, controlling prosthetics and treatment of disorders such as Parkinson's disease, epilepsy, and sensory loss^1,2. Measuring and/or controlling both the chemical and electrical composition of the brain is the basis for all neural implants. However, it is important to administer a treatment only when the neural tissue is in the aberrant state to reduce side effects³. For instance, deep brain stimulators for epilepsy treatment should only apply an electrical pulse to the brain during a seizure. Some side effects may be dystonia, loss of memory, disorientation, impaired cognitive function, induced hallucinations, depression or anti-depression^3,4. In many devices, a closed loop system is therefore necessary to record electrical activity and to trigger stimulation when an abnormal state is detected. Recording electrodes are also used to control prosthetic devices. It is critical to record the target neural activity with the highest possible signal-to-noise ratio to achieve the most accurate triggering and device control. A large signal-to-noise ratio is also highly desirable for research applications, as more reliable data can be obtained, resulting in fewer required test subjects. This will also allow a greater understanding of the mechanisms and pathways involved in neural stimulation and recording.

After a neural implant has been placed into the brain, an immune response is triggered^5,6. The time course of the response is generally divided into acute and chronic phases, each consisting of different biological processes⁷. The immune response can have dramatic effects on the performance of the implant, such as isolation of the electrodes from the target neurons by encapsulation in a glial scar or chemical degradation of the implant materials⁸. This can reduce the signal-to-noise ratio of a recording electrode and the power output of a stimulating electrode, and lead to electrode failure⁹. Careful choice of implant design and materials are necessary to prevent failure over the implant lifetime.

Many different materials and implant designs have been developed recently to improve the signal-to-noise ratio and implant stability for neural recording. Electrode materials have included platinum, iridium, tungsten, iridium oxide, tantalum oxide, graphene, carbon nanotubes, doped conducting polymers, and more recently hydrogels. Substrate materials tested also includes silicon, silicon oxide, silicon nitride, silk, Teflon, polyimide, and silicone.  Various electrode modifications have also been investigated, using coatings such as laminin, neurotrophins, or self-assembled monolayers and treatments using electrochemical, plasma and optical techniques. Implant designs could be 1-, 2- or 3-dimensional with the electrodes generally at the tip of an insulating probe or along the edge of a shank for penetrating electrodes or in a 2-dimensional array for cortex surface implants. Regardless of electrode design or material, previous literature has typically demonstrated the performance of the new implant without reference to other implant constructs. This prevents a systematic evaluation of their properties.

This protocol provides a method for comparing different electrode materials via a range of analytical and electrophysiological techniques. It is based on a recently published article which compared 4 different doped conducting polymer coatings (polypyrrole (Ppy) and poly-3,4-ethylenedioxythiophene (PEDOT) doped with sulfate (SO₄) or para-toluene sulfonate (pTS)) and 4 different coating thicknesses¹⁰. This article found one material, PEDOT-pTS with a 45 sec deposition time, had the highest signal-to-noise ratio and spike count with the smallest background noise and that these parameters were dependent on electrode impedance. PEDOT-pTS also displayed superior acute biostability compared to the other doped conducting polymers and bare iridium electrodes. The protocol allows the critical parameters controlling the signal-to-noise ratio and stability to be determined and used to further improve the performance of neural recording electrodes.

Protocol

The protocol has been approved by the La Trobe University (09-28P) and RMIT University Animal Ethics committees (1315).

1. Electrode Preparation and Preliminary in vitro Testing

1. Prepare electrode coating deposition solutions; for instance 10 mM 3,4-ethylenedioxythiophene (EDOT) and 0.1 M sodium para-toluene sulfonate (Na₂pTS) to form poly-3,4-ethylenedioxythiophene-pTS (PEDOT-pTS).
2. Connect the electrode array to a potentiostat.
3. Carefully place the electrode array into the deposition solution and clamp into place.
4. Place a platinum mesh counter electrode and Ag/AgCl reference electrode into the deposition solution and connect to a potentiostat.
5. Using the potentiostat, deposit coatings onto the desired electrodes. Deposition conditions (potential, current and time) will vary depending on the desired coatings. For PEDOT-pTS coatings, an applied potential of 1 V for 15, 30, 45, or 60 sec has been used.  Four electrodes on the array should be coated with the coating in a staggered configuration (Figure 1).
6. Remove the electrode array from the deposition solution and gently rinse with deionized water.
7. Repeat the coating procedure with other materials as desired.
8. Prepare in vitro testing solution (0.3 M di-sodium phosphate (Na₂HPO₄) in deionized water).
9. Connect the electrode array to a potentiostat.
10. Carefully place the electrode array into the testing solution and clamp into place.
11. Place a platinum mesh counter electrode and Ag/AgCl reference electrode into the testing solution and connect to a potentiostat.
12. Using the potentiostat, perform sequential electrochemical impedance spectroscopy (EIS) (potential offset 0 V, amplitude 10 mV, frequency range 10-100,000 Hz) and cyclic voltammetry (1 cycle, potential range 0.8 to -0.8 V, scan rate 100 mV/sec) on all electrodes. Untested electrodes are kept at open circuit potential and a quiet time of 1 sec is used between each test. All 32 electrodes are in contact with the solution for the full testing session of 1 hr.
13. Remove the electrode array from the testing solution and gently rinse with deionized water.
14. Perform any other desired analyses such as optical microscopy.
15. Store probes in a dry protective container to prevent damage and degradation of the electrode surfaces.

2. Electrode Implantation

1. Weigh the rat.
2. Inject urethane (20% w/v in distilled water, 1.3 g/kg i.p.) for nonrecovery anesthesia.
3. Ensure anesthesia onset by testing for a toe pinch withdrawal reflex. If anesthesia is not sufficient, supplementary doses of urethane should be administered (0.3 g/kg i.p.).
4. Apply eye lubricant, and then shave the head of the animal.
5. Place the animal in the prone position on a homeothermic plate and insert a rectal probe (37.5 °C).
6. Place one ear bar into the approximately expected final position within the stereotaxic frame, and then adjust the animal to position the ear bar in the external acoustic meatus.
7. Align the second ear bar into the contralateral external acoustic meatus. Shift the animal in the ear bars to align with the tooth holder.
8. Using rat-tooth forceps, open the animal's jaw, hook the upper incisors over the tooth holder and clamp the nose in place.
9. Create an incision in the skin of the head, approximately 1 mm to the right of the midline and from 10 mm rostral to 10 mm caudal of lambda.
10. Retract the skin and muscle laterally from the incision to expose the parietal and interparietal bones Using 20% hydrogen peroxide solution and a gauze pad, scrub the surface of the exposed bone.
11. Drill a hole approximately 3 mm² in the interparietal bone as close to lambda and the midline as possible and remove the bone plug. Using sterile saline, flush the hole to remove any bone dust or fragments which may damage the electrode.
12. Using blunt-blunt scissors, dissect below the scruff of the neck and create a cavity. Wrap a Ag/AgCl wire in cotton wool, saturate it with saline and then insert the reference electrode into the cavity.
13. Make an incision in the dura mater on the sagittal plane using the tip of a needle.
14. Attach the electrode array to the electrode manipulator and adjust its position over the opening with a 19° rostro-caudal angle. Manually insert the electrode approximately 2 mm into the brain towards the inferior colliculus.
15. Attach the speaker to the left hollow ear bar.
16. Ensure the amplifier is turned on. Then verify animal anesthesia before sealing the recording chamber.

3. In vivo Testing

1. Deliver white noise bursts, (Gaussian distributed noise, 1-44 kHz; 10 msec rise-fall time) and monitor the activity on each electrode. The maximum rate at which bursts should be delivered is one burst every 200 msec.
2. Using the motorized microdrive, slowly insert the electrode array until acoustically driven activity is recorded on the 3 most distal electrodes on each shank (the number and position of electrodes recording activity may vary with electrode placement or electrode design).
3. Perform the acoustic stimulation protocol using 300 repetitions of 50 msec white noise bursts (Gaussian distributed noise, 1-44 kHz; 10 msec rise-fall time) with a 1 sec repetition rate at 70 dB, and record the multiunit activity at each electrode (24.4 kHz sampling rate).
4. Slowly insert the electrode array another 200 μm into the IC to position each electrode in roughly the same position as the more distal electrode from the initial recording position.
5. Repeat the acoustic stimulation and neural recording protocol.
6. Continue inserting the electrode array in 200 μm steps and performing the acoustic stimulation and neural recording protocol until all electrodes have recorded acoustically driven activity from at least 3 positions (typically 8-12 electrode positions overall).
7. Retract the electrode array in 200 μm steps and continue performing the acoustic stimulation and neural recording protocol until the initial electrode array position is achieved.
8. Carefully retract the electrode array manually.
9. Inject an overdose of sodium pentobarbitone (Lethobarb; 200 mg/kg i.p.) to euthanize the animal.
10. Gently rinse the electrode array with distilled water.  Then store probes in a dry protective container to prevent damage and degradation of the electrode surfaces.

4. Post-implantation in vitro Testing

1. Gently rinse the electrode array with distilled water to remove any contamination.
2. Connect the electrode array to a potentiostat.
3. Carefully place the electrode array into the testing solution and clamp into place.
4. Place a platinum mesh counter electrode and Ag/AgCl reference electrode into the testing solution and connect to the potentiostat.
5. Using the potentiostat, perform sequential electrochemical impedance spectroscopy (EIS) (potential offset 0 V, amplitude 10 mV, frequency range 10-100,000 Hz) and cyclic voltammetry (1 cycle, potential range 0.8 to -0.8 V, scan rate 100 mV/sec) on all electrodes. Untested electrodes are kept at open circuit potential and a quiet time of 1 sec is used between each test.  All 32 electrodes are in contact with the solution for the full testing session of 1 hr.
6. Remove the electrode array from the testing solution and gently rinse with deionized water.
7. Place the electrode array into an enzymatic cleaning solution for 24 hr.
8. Remove the electrode array from the solution and rinse with distilled water.
9. Connect the electrode array to a potentiostat.
10. Carefully place the electrode array into the testing solution and clamp into place.
11. Place a platinum mesh counter electrode and Ag/AgCl reference electrode into the testing solution and connect to the potentiostat.
12. Using the potentiostat, perform sequential electrochemical impedance spectroscopy (EIS) (potential offset 0 V, amplitude 10 mV, frequency range 10-100,000 Hz) and cyclic voltammetry (1 cycle, potential range 0.8 to -0.8 V, scan rate 100 mV/sec) on all electrodes. Untested electrodes are kept at open circuit potential and a quiet time of 1 sec is used between each test.  All 32 electrodes are in contact with the solution for the full testing session of 1 hr.
13. Remove the electrode array from the testing solution and gently rinse with deionized water.
14. Store probes in a dry protective container to prevent damage and degradation of the electrode surfaces.

Results

A typical electrode array used for this experimental protocol is shown in Figure 1. There are 32 iridium electrodes on 4 shanks with 413 μm² nominal geometric area and a 200 μm pitch. Every second electrode on the array has been coated with one of four different electrode coatings, labeled 1-4. The coating materials have been carefully chosen for their chemical, mechanical and electrochemical properties. As mentioned previously¹⁰, increased deposition times will in...

Discussion

This protocol provides a method for comparing neural recording electrode coatings within one animal. The electrode design used is ideal for implantation into a rat inferior colliculus (IC), with dimensions of a similar scale. Variations of this electrode such as more space between shanks would prevent all shanks being in the rat IC at the same time, while longer shanks and a larger pitch between electrodes increase the risk that the shank tips will come in contact with the base of the skull during insertion.
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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Programmable Attenuator	TDT	PA5	Controls the amplitude of the acoustic signal across frequencies
Electrostatic speaker driver	TDT	ED1	Drives the electrostatic speakers (EC1)
Coupled electrostatic speaker	TDT	EC1	Delivers sound to the animal
Processing base station	TDT	RZ2	Records neural activity from electrode array (using PZ2 preamplifier)
Preamplifier	TDT	PZ2-256	256-channel high impedance preamplifier
Multifunction Processor	TDT	RX6	Used to generate acoustic stimuli
Multichannel electrode	NeuroNexus Technologies	A4 × 8–5mm-200-200-413	4-shank 32-channel electrode array
Potentiostat	CH Instruments	CHI660B	Deposits electrode coatings and performs cyclic voltammetry and EIS (used with CHI684)
Multiplexer	CH Instruments	CHI684	Switches between electrodes on the potentiostat
Disodium phosphate	Fluka	71644	Used in the test solution
3,4-Ethylenedioxythiophene (EDOT)	Sigma Aldrich	483028	An electrode coating material
para-Toluene sulfonate (Na2pTS)	Sigma Aldrich	152536	An electrode coating material
Urethane	Sigma Aldrich	U2500	Used to anesthetize the animal
Silver/Silver chloride electrode	CH Instruments	CHI111	Used for testing the electrode in vitro
Platinum electrode	CH Instruments	MW4130	Used for testing the electrode in vitro
Motorized microdrive	Sutter Instruments	DR1000	To control the electrode array position during surgery
Enzymatic cleaner	Advanced Medical Optics	Ultrazyme	Cleans the protein off the electrode array after implantation
Acoustic enclosure	TMC Ametek	83-501	Isolates the animal from acoustic and electrical noise
Stereotaxic frame	David Kopf Instruments	1430	Secures and positions the animal
Temperature controller	World Precision Instruments	ATC1000	Controls the animal temperature
Bone drill	KaVo Dental	K5Plus	Used to perform the craniectomy
Aspirator	Flaem	Suction pro	Used to perform the craniectomy