The Evolution of the Optrode

The Evolution of the Optrode:

An Overview of in vivo Neuronal Modulation and Recording

Mackenzie M. Andrews, Isaac Clark, Uri Zvi

University of Washington, Bioengineering

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Abstract

A primary goal in neuroscience research is to understand the activity of individual neuronal populations within a

specific behavioral context. This goal is presented with challenges such as the heterogeneity of cell types within

the brain as well as the high speed (millisecond timescale) of brain activity. In addition, animal models are

needed for high throughput, controlled studies of brain activity. Many researchers use mouse models for

behavioral research, but mice present additional constraints due to their small size. To confront these challenges,

researchers have combined genetic methods with small scale engineering to develop tools to specifically target

and record from neuronal populations within awake and behaving mice. The combined use of optical

modulation and electrode recording has coined the term ‘optrode’ within the field. This paper addresses the

history, evolution, and potential future directions of the optrode.

Keywords: optrode, optogenetics, electrophysiology, neural engineering

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The Evolution of the Optrode:

An Overview of in vivo Neuronal Modulation and Recording

Introduction

In recent studies researching neural signals, the use of in vivo electrophysiology probes has become

more prevalent. These probes allow for the very precise monitoring of individual neurons. The use of multiple

electrophysiology wires or multi-array silicon probes can also allow for simultaneous monitoring of numerous

individual neurons. This can be extremely beneficial when the precise location of a phenomenon in the brain is

already known, but data on the activity in that location is needed. For example, if a neuron has been seen to

activate in response to the subject viewing the color green, electrophysiology probes may be beneficial to

determine the effects on that specific neuron while viewing different intensities of green.

Through the use of optogenetics, certain proteins that are responsive to light can be genetically inserted

into cells. The presence of these proteins can be used to modulate various cellular functions such as excitation or

inhibition of action potentials. The neurons can then be either stimulated to initiate an action potential or

inhibited when exposed to certain wavelengths of light. The light exposure is often delivered by the controlled

use of optical fibers. Two of the primary proteins used in neuroscience today are Channelrhodopsin-2 (ChR2)

and halorhodopsin (NpHR). These were discovered to have an open channel effect when exposed to certain

wavelengths of light. ChR2, opens when exposed to blue light and allows the flow of ions which, depolarize the

cell and stimulate an action potential. NpHR will conversely open when exposed to yellow light and will allow

the flow of ions that hyperpolarize the cell and inhibit action potentials. Due to their difference in wavelength

trigger activation they are often used in tandem. This practice can be used in research to determine the effects of

the stimulation as well as to treat certain diseases if the effect is already known.

Due to the heterogeneity of neuronal cell types, we can use cell targeting abilities of optogenetics to

introduce these protein to specific cells in the brain. This is often done via cell type specific promoter fragments

that are introduced into to the subject or specified breeding. They are often introduced into the cells via viral

vectors that carry the promoters. These promoters are then read many times and the corresponding proteins are

produced and integrated into the cell.

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The combination of electrophysiology probes, optogenetics, and optical fibers can be used to stimulate

and monitor that stimulation in a specific neuron or group of neurons. The controlled perturbation of local

circuits allows the researcher to gain a better understanding of how the circuit is connected. With that

information, treatments can then be formed or improved based upon it.

Paper 1

Targeting and Readout Strategies for Fast Optical Neural Control In Vitro and In Vivo

As previously mentioned, the heterogeneity of the neuronal cells is a major reason why optogenetics

have become so useful. In 2007, Gradinaru et al. wrote a review on the first strategies combining optogenetic

modulation with electrophysiological recording. They described that optogenetics allow for the targeting of cells

in various ways such as viral mediated injection during development to incorporate the proteins into neural

layers, injection with promoter fragments that only promote in specific subtypes of neurons or specific

subcellular portions of the neurons, and also the simple projection of the photo stimulus. All of these targeting

methods allow for more specified research from locations and subtypes of cells even to the subcellular level of a

single neuron. The group showed examples of research that had been done that shows these methods were all

quite effective in different ways (Figure 1).

The group described that the implementation of optically stimulated proteins can be paired with

numerous methods of data recording. A major benefit being the fact that optically stimulating a neuron will not

produce any electrical artifacts. It can be paired quite effectively with physio electrical signal recording done via

a simple electrode. Due to the high speed of these signals and effective recording, very high temporal resolution

can be seen. Optical neural control can also be paired with other methods such as luminescent optical recording

and long term biochemical analysis. If the speed of acquisition and optical stimulation delivery is sufficient,

luminescent optical recording can be used to determine the molecular and ionic information concerning neuronal

and local circuit action potentials. The long term biochemical analysis on the other hand, may allow for the

long-term tracking and validation of the effects optical neural control has on neurons and circuits. Through

simple visual analysis however, larger scale changes can be seen through the use of optical neural control.

Specifically, the behavior of conscious rodents can be altered via optogenetic stimulation. This could be seen in
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an experiment in which, only the right side of a rodent's brain was optogenetically stimulated while awake. This

experiment resulted in the continued tendency for the rodent to turn left and walk in a counterclockwise spiral

while stimulated (Figure 2).

Paper 2

Multi-array silicon probes with integrated optical fibers: light-assisted perturbation and recording of local

neural circuits in the behaving animal

Once the idea of the optrode was exposed to the field, there was a major push to increase the spatial

resolution of the devices. In order to achieve this aim, Royer et al., (2010) developed multi-array silicon probes

equipped with micron-scale optical fibers for optogenetic probing of neural circuits. Figure 3 shows the general

engineering design and images of the completed device. The group etched the optical fiber using hydrofluoric

acid and then cured the fiber to the silicon probe via UV curable epoxy (Figure 3A-B). The optical fiber

equipped shanks were then arranged in either 4 or 8 shank probes (Figure 4). The shanks were designed such

that the optical fiber could terminate between 300 micrometers before the tapering of the shank to 100

micrometers past the end of the shank, allowing for versatility in where the light modulation was occurring.

Using these multi-shank silicon probes, the group injected a parvalbumin targeting

halorhodopsin virus into the hippocampus of mice. By implanting a 4-shank probe with one light equipped

shank into the halorhodopsin expressing animals, the group was able to show spatial specificity in neural

modulation via the light equipped channel (Figure 5). The 8-shank probe was capable of recording from “50 to

140 well-clustered neurons,” which was a significant increase in spatial resolution compared to previous

methods. Using this technique, the group went on to show an example of a behavioral task in which light

modulation was applied, selectively activating specific neurons during the task (Figure 6).

Paper 3

The flexDrive: an ultra-light implant for optical control and highly parallel chronic recording of neuronal

ensembles in freely moving

Chronic recording of neuronal ensembles is challenging, especially in behaving mice. The complexity

of available drive mechanisms, implant weight tolerated by mice, and finding the precise positioning for high-
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quality recording, limits current methods to recordings from no more than 4–8 electrodes in a single target area.

This becomes even more complicated when integrating optic fibers for optogenetic manipulations. Mice comfort

and survivability imposes weight limit of about 4g, and even 2g when a combination of recording with

behavioral phenotyping is desired (motion and videography needs). Previous studies bypassed the weight

problem by offsetting the implant weight with a pulley system or attaching a helium-filled balloon to the

implant, or by placing the animal on a headpost, which aren’t ideal. Another method utilizes static electrode

arrays to make large-scale recordings within a lightweight device. However, in case of inaccurate positioning or

glial encapsulation, use of static electrodes prevents an easy change in position. There is a need for a low

weight, with high number of individually movable electrodes, high placement stability, and independently

adjustable optical fibers that would allow high-quality recording in behaving mice.

A highly miniaturized drive design that replaces the drive mechanisms found in current implants with a

one-piece spring design was developed. “FlexDrive” fits 16 individually movable tetrodes (up to 64 channels),

can maintain stable recording conditions for months, integrates 2+ optic fibers, and only weighs ~2g (Figure

7B). Electrodes are positioned by an array of flexible polyimide guide tubes, while the electrode sits in a

movable “shuttle tube” that can be laterally adjusted using screws (Figure 7A), where each screw turn

corresponds to a 250μm lateral motion. This allows the user to adjust the position of individual electrode, even

after surgery, which is important for several reasons like in the case of glial encapsulation.

An injection of adenovirus was used to deliver the gene for channelrhodopsin-2 (ChR2) to the SI region

and the device was mounted, all in the same surgical procedure (Figure 8A). The authors were able to show

recorded activity from relevant neurons in response to laser pulses (Figure 8B). In addition, a multi-site

recording from both the SI region and the thalamic reticular nucleus (RTN) was achieved by adjusting

individual electrodes, showing that the design can produce parallel recordings from 16 electrodes from two sites

with the accuracy required to observe neurons in small, deep targets such as the TRN. To conclude, the

flexDrive presents a straightforward method for obtaining stable and high-quality electrophysiological data from

multiple target sites in awake, behaving mice. This permits researchers to make full use of the precision and
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specificity of optogenetic methods by directly probing the concerted function of neural circuits, rather than

individual neurons.

Paper 4

Fully integrated silicon probes for high-density recording of neural activity

Continuing on the trend of increasing spatial resolution during neural recording, Jun et al. (2017) took

the silicon probe to a new level of spatial specificity. With the goal of high spatial resolution across a large

recording area, the group fabricated a silicon probe, which they coined ‘Neuropixels.’ The probe incorporated

384 recording channels capable of programmably addressing 960 individual recording sites along the 10-mm

long, 70×20-micrometer cross-section shank (Figure 9). The probe was fabricated and programmed similar to

computer chips and allows for electrical recording across large neuronal populations and multiple brain regions.

In awake, head fixed mice, the group was able to implant two probes and record from more than 700

well-isolated single neurons within 5 brain structures (Figure 10). Each probe is capable of recording from

approximately 300 neurons at a time. This has only been accomplished with a 16 shank silicon probe similar to

those described in the second paper (Royer et al., 2010). Thus, the Neuropixels probe drastically improves

spatial resolution while decreasing the size of the implant.

The group also showed that the probe was compatible with optical excitation conditions used for

optogenetic modulation. In a saline solution, the probe showed little recording artifact due to direct illumination

suggesting that the probe would be viable for in vivo recording in tandem with optical stimulation. Preliminary

data from such recordings suggests that the Neuropixels silicon array may be a glimpse at the future of the

optrode.

Future Directions

The Neuropixels silicone probe, described in James J. Jun et al., constitutes a real advancement when it

comes to spatiotemporal resolution and coverage of large volume. However, the paper is lacking two obvious

components that were present in other papers featured in this review. First, it would be interesting and necessary

to examine the results of these superior neuronal recordings when modulated by optogenetics in behaving mice.

Then it would be possible to investigate whether the new data allows us to better control/predict/ identify free
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behaviors in mice. Second, the Neuropixels lack the ability for adjusting individual electrode post-surgery. A

device with comparable spatiotemporal resolution that would have such an ability would be superior since it

won’t be susceptible to glial encapsulation.

Another interesting future research direction is the ability to incorporate multi-site recordings for

investigation of coordination dynamics. The “flexDrive”, featured here and designed by Voigts et al., showed

limited potential for multi-site recording, but it is very restricted in application and comes on the expense of

spatial resolution (less electrodes for each site). A device that would adhere to the weight and size constrictions,

but would allow high resolution, multi-site recording via, for example, two different drivers, would be a nice

step forward in our ability to investigate multi-site coordination dynamics.

Lastly, we want to jump many years forward and discuss possible clinical applications. Even though we

still need to translate these designs to larger animals, and eventually humans, when we finally achieve that goal

and implant such an optrode-based device in humans, the exciting possibilities seem endless. One example

would be the trend that is currently being investigated in almost all monitoring fields- a closed-loop therapeutic

system. Such a system would be able to predict upcoming adverse events (an epileptic attack, a stroke, etc.) and

apply the needed neuronal stimulation to prevent it or substantially ease the symptoms.

References

Gradinaru, V., Thompson,K.R., Zhang, F., Mogri, M., Kay, K., Schneider, M.B., & Deisseroth, K.

(2007). Targeting and Read out Strategies for Fast Optical Neural Control In Vitro and In Vivo. J. Neurosci.

27(52):14231–14238.

Jun, J.J., et al. (2017). Fully integrated silicon probes for high-density recording of neural

activity.Nature. 551(7679):232-236.

Royer,, S., Zemelman, B.V., Barbic, M., Losonczy, A., Buzsáki, G., & Magee, J.C. (2010). Multi-array

silicon probes with integrated optical fibers: light assisted perturbation and recording of local neural circuits in

the behaving animal. Eur J Neurosci. 31(12): 2279–2291.

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Voigts, J., Siegle, J.H., Pritchett, D.L. & Moore, C.I. (2013). The flexDrive: an ultra-light implant for

optical control and highly parallel chronic recording of neuronal ensembles in freely moving mice. Front. Syst.

Neurosci. 7(8): 1662-5137.

Figures

Figure 1: Optogenetic Targeting Techniques - Immunohistochemical validation of a number of opsin targeting

modalities including through breeding (A), via axonal projection (B), viral promoter targeting (C), and
subcellular targeting (D). (Gradinaru et al., 2007)

Figure 2: Optical Control of Behavior – Walking behavior of a mouse before (green), during (black), and after
(red) optical stimulation. Optical control of the right motor cortex caused the animal to walk in left hand circles.
(Gradinaru et al., 2007)
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Figure 3: Silicon Array Schematic – A-top) Etching of optical fiber via hydrofluoric acid; B) Securing of
optical fiber to silicon array via UV curable epoxy; C) Images of completed silicon array; A-bottom) Schematic
of implantation into a rodent. (Royer et al., 2010)

Figure 4: Multi-Shank Silicon Probe – A) 4-shank probe with one light equipped channel; B) 8-shank silicon
probe with 4 light equipped channels; C) 8-shank silicon probe with 4 light equipped channels of variable fiber
optic termination location. (Royer et al., 2010)

Figure 5: Light Modulation – Sinusoidal light modulation on one channel of a 4-shank probe showing spatial
specificity in neural activity modulation. (Royer et al., 2010)
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Figure 6: Neural Modulation During Behavior – A) Behavioral set up; B) Light modulation during specific
behavioral region; C) Recordings from 10 cells during behavior showing specificity inn responsiveness. (Royer
et al., 2010)

Figure 7: flexDrive Schematic - A) Internal view of flexDrive featuring the interface board, the 16 adjusting
screws, the polyimide drive body, and the drive spring; B) External view of the flexDrive mounted on a mouse’s
head, weighing only ~2g and 22mm head to cap; C) Adjustable electrode mechanism. The mobile shuttle tube is
adjustable by the drive spring (connected to the screw) and slides laterally inside the guiding tube to control the
position of the electrode. (Voigts et al., 2013)

Figure 8: Optical Modulation with flexDrive - A) Activation of neurons in S1 region with ChR2; B) Trace of an
identified neuron on one of the tetrodes for one session. (Voigts et al., 2013)
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Figure 9: Fully Integrates Silicon Array Schematic – Silicon array containing 960 recording sites with 384
live channels. (Jun et al., 2017)

Figure 10: In Vivo Recording with Fully Integrated Array – A) Probe 1 site and isolated unknit recording from
3 brain regions; B) Probe 2 site and isolated unknit recording from 2 brain regions. (Jun et al., 2017)