A novel carbon tipped single micro-optrode for combined optogenetics and electrophysiology

Abstract

Optical microelectrodes (optrodes) are used in neuroscience to transmit light into the brain of a genetically modified animal to evoke and record electrical activity from light-sensitive neurons. Our novel micro-optrode solution integrates a light-transmitting 125 micrometer optical fiber and a 9 micrometer carbon monofilament to form an electrical lead element, which is contained in a borosilicate glass sheathing coaxial arrangement ending with a micrometer-sized carbon tip. This novel unit design is stiff and slender enough to be used for targeting deep brain areas, and may cause less tissue damage compared with previous models. The center-positioned carbon fiber is less prone to light-induced artifacts than side-lit metal microelectrodes previously presented. The carbon tip is capable of not only recording electrical signals of neuronal origin but can also provide valuable surface area for electron transfer, which is essential in electrochemical (voltammetry, amperometry) or microbiosensor applications. We present details of design and manufacture as well as operational examples of the newly developed single micro-optrode, which includes assessments of 1) carbon tip length-impedance relationship, 2) light transmission capabilities, 3) photoelectric artifacts in carbon fibers, 4) responses to dopamine using fast-scan cyclic voltammetry in vivo, and 5) optogenetic stimulation and spike or local field potential recording from the rat brain transfected with channelrhodopsin-2. With this work, we demonstrate that our novel carbon tipped single micro-optrode may open up new avenues for use in optogenetic stimulation when needing to be combined with extracellular recording, electrochemical, or microbiosensor measurements performed on a millisecond basis.

Fig 1. The carbon tipped coaxial single micro-optrode.

(A) Overall view illustrating major components. Light was delivered through the optical fiber and conducted further by the borosilicate glass extension light guide. Electrical signals picked up by the carbon tip were transmitted to amplifiers by the gold-plated miniature pin built in the plastic holder. (B) A three-dimensional rendering illustrating the construction of the apex. The solid glass light guide also served as a mechanical support and tightly sealing electrical insulator. (Bb) Scanning electron microscopic image of the final glass taper segment with a protruding 100 μm long sharpened carbon tip. (C) Light microscopic image of the optical fiber end, the light guiding glass taper and center-positioned carbon fiber extending from the dome-shaped glass ending.

Fig 2. Scanning electron micrographs showing tip structure of micro-optrodes.

(A, B) View of a ground tip demonstrating the center position carbon disk surrounded by an annulus of glass. The glass served as mechanical support, an electrical insulator, and a light guide. (C, D) Endings of intentionally broken tips exposing tight junctions between carbon fiber and glass sheathings. (E, F) The surface roughness of a carbon tip revealing the effects of spark etching on a pitch-type carbon fiber. Note the longitudinal bundles of carbon fiber. See also panel D.

Fig 3. Impedance of the carbon tip as a function of length.

The curve was best fitted with an extended Langmuir adsorption isotherm equation. Mean impedances ± SD of n carbon tips for each length were determined in saline at 1 kHz and plotted against lengths as shown. Impedances of ground-tip carbon disks were also determined and their lengths were taken as 1 μm. Scanning electron micrographs of 25 μm and 100 μm long carbon tips are shown as insets A and B, respectively.

Fig 4. Light projection power through the tip as a function of light source power.

Light was delivered through the built-in optical fiber connecting to the 473 nm laser diode light source. Light power projected by optrode tips and detected with a flat sensor surface in air were measured using ground or dome-shaped tips. Mean ± SD of n micro-optrodes were calculated and shown.

Fig 5. Photoelectric effects in tungsten and carbon tip microelectrodes.

Light-induced potentials were recorded from laterally illuminated microelectrodes as illustrated on the left. Amplitudes (A) of the initial phase of the artifacts were measured as shown by the sample recordings at the top of the right panel. Data are presented as mean ± SD. All comparisons were significant (P < 0.01).

Fig 6. Photoelectric effects in carbon tipped micro-optrodes.

Upon illumination through the built-in optical fiber, the photoelectric artifacts in the carbon fiber were dependent on light power and the lengths of the uninsulated carbon tips. Recordings were made as shown on the left and amplitudes (A) of the initial phase of the artifacts were measured as indicated on the sample traces in the middle. Data are presented as mean ± SD. Note the complete lack of artifacts in the ground-tip micro-optrodes.

Fig 7. Optogenetic stimulation and extracellular recordings with a 25 μm carbon tip.

Sample recordings were taken from the hippocampus of the rat brain transfected with channelrhodopsin-2 using a dome-shaped glass tip for light projection with a 25 μm carbon tip as recording element. Light stimuli were delivered at 473 nm from a 70 mW laser source in continuous epochs, as shown by horizontal bars. (A, B) Light-sensitive neurons were either excited or, in a smaller number, inhibited during delivery of light. (C, D) Local field potentials were recorded with filter settings that ranged from 1 Hz to 150 Hz. Segments designated in panel B were detailed showing the recorded traces around the onset (C) and conclusion (D) of the light stimulation. No noticeable photoelectric artifacts were recorded under these conditions.

Fig 8. Voltammetric responses of carbon tips to DA.

Average peak fast-scan cyclic voltammetry oxidation currents plotted against the concentration of dopamine (DA) dissolved in phosphate-buffered saline. Values represent the mean ± SD of six micro-optrodes with 100 μm carbon tips. (Top left inset) Visualization of DA-evoked oxidation currents as a function of the electrode potential and time using the Demon Voltammetry and Analysis Software. (Bottom right inset) A representative background-subtracted cyclic voltammogram recorded at 1 μM DA concentration.

Fig 9. Stimulation-evoked oxidation currents in the nucleus accumbens.

A representative experiment exemplifying the performance of a micro-optrode with 100 μm carbon tip in fast-scan cyclic voltammetric (FSCV) determination of peak oxidation currents in the nucleus accumbens in response to electrical stimulation of the ventral tegmental area (VTA). The prenomifensine peak current was taken as control. The maximum oxidation current was measured 21 minutes postnomifensine intraperitoneal administration. The inset in the middle shows the sites of electrical stimulation and FSCV recordings in a silhouette of the rat brain. Traces of actual voltammograms are shown in the lower right inset.