BlueBerry: Closed-loop wireless optogenetic manipulation in freely moving animals

Reviewer #1 (Public review):

Summary:

This paper presents a wireless device for closed-loop control of optogenetic stimulation based on behavioral triggers. The authors demonstrate the device through two behavioral experiments in mice, showcasing the device's capabilities and emphasizing open accessibility and using off-the-shelf components.

Strengths:

The paper presents a device that is open access and easily reproducible for wireless stimulation in a closed loop based on behavioral triggers. Other strengths of the device include the simultaneous use of multiple devices in parallel and the claimed ease of integration with existing frameworks. The paper shows to behavioral experiments on multiple mice along with some device validation results.

Weaknesses:

The main weakness of the presented device lies in the lack of flexibility in stimulation power. For a device that is intended for stimulation only, having to physically change a component on the board to adapt stimulation power is a major downside. Reprogrammable stimulation current is not complex to implement and should really have been included on this device. Another weakness lies in the limited battery life of the device. While using a battery-powered device decreases spatial constraints, allowing for the maze experiment presented in the paper, it also means the lifespan of the device is limited compared to an inductively powered device, limiting its ability for long-term experiments.

Reviewer #2 (Public review):

Summary:

The authors have developed an elegant, lightweight, open-source system that should be able to be widely disseminated to the community. They have used this system in multiple experimental paradigms and demonstrate its functionality quite elegantly. One of these experiments involves two of three animals in the arena being stimulated, a situation that clearly requires an untethered approach. They have appropriately quantified key system parameters (latency and battery life).

Strengths:

The introduction places this work in a broader context. That context includes a number of previous solutions, many of which are smaller or more technically complex. However, I agree with the authors that there is a need for something that is easy for labs to acquire and deploy in terms of both what goes on the head and the broader infrastructure (i.e., not needing complex wireless power delivery approaches).

The paper does an excellent job of describing the system architecture. And the architecture is good! Their system comprises more than just the bluetooth enabled head-mounted devices - they also have built an interface that allows for TTL triggers that link into existing workflows.

The key metrics for a device like this are weight, battery life, and latency. The weight is 1.4g, which is appropriate for adult mice; the battery life is ~100 minutes of continuous stimulation, which should be sufficient for many experiments, and the latency is typically less than 30 ms, which is fine for all but the most demanding closed-loop experiments.

Performance is demonstrated in two experiments, a continuous Y-maze, which elegantly demonstrates how transfected animals learn to sense optogenetic closed-loop stimulation to drive their choice behavior in a way that control-stimulated animals do not. While authors claim that the ~2m diameter apparatus is "large scale", the second behavior more convincingly demonstrates the need for wireless stimulation.

They used closed-loop monitoring of animal pose to selectively stimulate animals for approaching the tails of a dominant conspecific (based on pre-experimental pairwise assessments). It seems that the original hope was that the increases in following that they observe would result in long-lasting changes in the hierarchy of a cage, but as they report, this was not observed. Critically, their supplementary video demonstrates that they conducted this experiment with two instrumented animals simultaneously. This is a situation where a tether would have been hopelessly tangled within a few moments!

The online documentation seems complete, and it seems quite possible for other labs to adopt and deploy the system.

Weaknesses:

The battery life is highly dependent on the stimulation paradigm. It makes sense that the LED is a major component of power consumption. It would have been elegant to measure the total optical energy that can be provided by the system. In addition, Bluetooth transmission is probably a major consumer of power, and receiving may not be "free". Quantifying power as a function of Bluetooth message rates would have been useful.

Presumably, the major constraint on latency is that the Bluetooth receiver polls at ~10 Hz, resulting in latency blocks of 20+, 30+, or 40+ ms. Why latency is never less than 10 ms is unclear. Could latency be reduced by changing a setting? Having a low-latency option would be very helpful for some experimental situations. Latency is probably the primary weakness of the system.

The programming process sounds quite complicated. It would be nice if they had OTA updates. But described and open source. Similarly, the configuration process (Arduino IDE) seems a bit complex. It would be nice if there were a dedicated cross-platform application.

It is unclear what the maximum number of devices that could be used without wireless interference is. The base station has two charging stations, but it would have been nice to understand the limits beyond this number.

There is a very nice website for the system, but there is some concern that the code and design files are not archived. Could they be deposited with the paper?

Reviewer #3 (Public review):

Summary:

This study presents a novel device for wireless control of optogenetic stimulation of the mouse brain, the Blueberry, using Bluetooth Low Energy (BLE) communication for parallel activation of up to 4 devices through an Arduino interface. The authors also present two types of brain implants for light delivery that can be connected to the Blueberry: one using uLEDs for surface cortical stimulation, and another using optical fibers for intra- or sub-cortical implants. The architecture of the system, including electronics, communication, and programming, is thoroughly described. Because the system was especially designed to be integrated with existing software used for neuroscience behavioral experiment for closed-loop experiments, validation of the system is shown on two different scenarios: a learning task in a "infinite" Y-maze, where light delivery at precise locations conditions arm choice for navigation; and a social interaction analysis where 3 animals are simultaneously stimulated in order to alter social dynamics among the group.

Strengths:

(1) The full system can be built by individual labs with simple PCB printing, off-the-shelf components, and readily available hardware (Arduino) for widespread dissemination.

(2) Four headstages can be controlled in parallel for simultaneous experiments with multiple mice.

(3) Validation across different relevant behavioral tests, demonstrating the potential of integrating Bluberry in closed-loop setups.

Weaknesses:

(1) Some details in the manuscript regarding system characterization (latency, battery life, etc) are included only in the supplementary materials.

(2) The practical details of integration with other commercial and open-source software used for the closed-loop experiments, which could help third-party researchers interested in using the system, are lacking sufficient detail.

(3) System range (3 meters reported) is limited for a BLE device.

(4) Light output amplitude is not programmable, limiting the choice of stimulation protocols and LEDs used.

(5) Thermal modeling of the cortical surface stimulator was not performed, and it is unclear if the brain implant for this purpose is within the safety limits.

(6) The paper is missing a comparison with other state-of-the-art devices for wireless control of optogenetic stimulation in mice.

Author response:

eLife Assessment

This work presents a valuable new open-source tool for wirelessly controlling optogenetic stimulation in neuroscience experiments in behaving rodents. Evidence for its potential usefulness in different types of optogenetic experiments is solid, although some details and concerns were viewed as lacking or overlooked (e.g., system latency, battery weight). The work is expected to interest neuroscientists working with optogenetics and neuroengineers developing small-sized integrated devices for rodent experiments.

We thank the eLife team for taking the time to consider and assess our manuscript. Please find below our provisional author responses accompanying the first version of the Reviewed Preprint.

We would like to clarify an important error regarding the battery model reported in the manuscript. We mistakenly referred to the CP1254-A3 (1.8 g), whereas the battery used for all devices is the CP9440 A4X (0.8 g).

Importantly, this correction reduces the total device weight by approximately 1 g compared to the value assumed by Reviewer #3. We believe this directly addresses the concern raised regarding battery weight in both the individual review and the overall eLife assessment.

We will correct this error in the revised manuscript and clearly report the exact battery model and total device weight.

For reference, the official VARTA CoinPower catalog is available here:

https://www.varta-ag.com/fileadmin/varta/industry/downloads/products/lithium-ion-cells/VARTA_CoinPower_EN_digital_221124_A5_6p.pdf

The battery used in BlueBerry is listed on the last line of page 2.

Public Reviews:

Reviewer #1 (Public review):

Summary:

This paper presents a wireless device for closed-loop control of optogenetic stimulation based on behavioral triggers. The authors demonstrate the device through two behavioral experiments in mice, showcasing the device's capabilities and emphasizing open accessibility and using off-the-shelf components.

Strengths:

The paper presents a device that is open access and easily reproducible for wireless stimulation in a closed loop based on behavioral triggers. Other strengths of the device include the simultaneous use of multiple devices in parallel and the claimed ease of integration with existing frameworks. The paper shows to behavioral experiments on multiple mice along with some device validation results.

We thank the reviewer for the statement.

Weaknesses:

The main weakness of the presented device lies in the lack of flexibility in stimulation power. For a device that is intended for stimulation only, having to physically change a component on the board to adapt stimulation power is a major downside. Reprogrammable stimulation current is not complex to implement and should really have been included on this device. Another weakness lies in the limited battery life of the device. While using a battery-powered device decreases spatial constraints, allowing for the maze experiment presented in the paper, it also means the lifespan of the device is limited compared to an inductively powered device, limiting its ability for long-term experiments.

We thank the reviewer for these valuable comments. We did consider implementing programmable control of stimulation power, for example using a digital potentiometer. However, in our current design this approach was not sufficient because the output current supported by typical digital potentiometers is too low for the high-power LEDs used in our system. For this reason, we did not include programmable stimulation current in the present version. We agree that this is a limitation and that further work is needed to identify a suitable solution for adjustable stimulation power, which we plan to pursue in future versions of the device. We will revise the manuscript to make this limitation and future direction clearer.

We also agree that the use of a battery-powered wireless system introduces an important trade-off. We will revise the manuscript to discuss this limitation more explicitly.

Reviewer #2 (Public review):

Summary:

The authors have developed an elegant, lightweight, open-source system that should be able to be widely disseminated to the community. They have used this system in multiple experimental paradigms and demonstrate its functionality quite elegantly. One of these experiments involves two of three animals in the arena being stimulated, a situation that clearly requires an untethered approach. They have appropriately quantified key system parameters (latency and battery life).

Strengths:

The introduction places this work in a broader context. That context includes a number of previous solutions, many of which are smaller or more technically complex. However, I agree with the authors that there is a need for something that is easy for labs to acquire and deploy in terms of both what goes on the head and the broader infrastructure (i.e., not needing complex wireless power delivery approaches).

The paper does an excellent job of describing the system architecture. And the architecture is good! Their system comprises more than just the bluetooth enabled head-mounted devices - they also have built an interface that allows for TTL triggers that link into existing workflows.

The key metrics for a device like this are weight, battery life, and latency. The weight is 1.4g, which is appropriate for adult mice; the battery life is ~100 minutes of continuous stimulation, which should be sufficient for many experiments, and the latency is typically less than 30 ms, which is fine for all but the most demanding closed-loop experiments.

Performance is demonstrated in two experiments, a continuous Y-maze, which elegantly demonstrates how transfected animals learn to sense optogenetic closed-loop stimulation to drive their choice behavior in a way that control-stimulated animals do not. While authors claim that the ~2m diameter apparatus is "large scale", the second behavior more convincingly demonstrates the need for wireless stimulation.

They used closed-loop monitoring of animal pose to selectively stimulate animals for approaching the tails of a dominant conspecific (based on pre-experimental pairwise assessments). It seems that the original hope was that the increases in following that they observe would result in long-lasting changes in the hierarchy of a cage, but as they report, this was not observed. Critically, their supplementary video demonstrates that they conducted this experiment with two instrumented animals simultaneously. This is a situation where a tether would have been hopelessly tangled within a few moments!

The online documentation seems complete, and it seems quite possible for other labs to adopt and deploy the system.

We appreciate the reviewer’s enthusiasm. Thank you.

Weaknesses:

The battery life is highly dependent on the stimulation paradigm. It makes sense that the LED is a major component of power consumption. It would have been elegant to measure the total optical energy that can be provided by the system. In addition, Bluetooth transmission is probably a major consumer of power, and receiving may not be "free". Quantifying power as a function of Bluetooth message rates would have been useful.

We thank the reviewer for this important suggestion. We agree that this is a missing characterization in the current manuscript. In the revised version, we will include a more detailed analysis of the system’s power budget, including the maximum stimulation power supported by the BlueBerry device, the corresponding output currents, and the contribution of the main integrated circuits to overall current consumption.

Presumably, the major constraint on latency is that the Bluetooth receiver polls at ~10 Hz, resulting in latency blocks of 20+, 30+, or 40+ ms. Why latency is never less than 10 ms is unclear. Could latency be reduced by changing a setting? Having a low-latency option would be very helpful for some experimental situations. Latency is probably the primary weakness of the system.

In the revised manuscript, we will clarify more explicitly that latency is a key limitation of the current system. We will also further investigate the source of this latency, including whether it can be reduced through additional configuration changes. In addition, we will include comparative latency measurements using different Arduino modules as the central BLE controller for the BlueHub device.

The programming process sounds quite complicated. It would be nice if they had OTA updates. But described and open source. Similarly, the configuration process (Arduino IDE) seems a bit complex. It would be nice if there were a dedicated cross-platform application.

We will investigate this matter and provide a simpler install and configuration script to setup both the BlueHub and Blueberry systems.

It is unclear what the maximum number of devices that could be used without wireless interference is. The base station has two charging stations, but it would have been nice to understand the limits beyond this number.

Due to the current structure of the ArduinoBLE library used in BlueHub devices, each BlueHub unit can support active communication with up to maximum 3 BlueBerry units. We thank the reviewer for highlighting this point and in the next version of the paper we will clarify this point.

There is a very nice website for the system, but there is some concern that the code and design files are not archived. Could they be deposited with the paper?

In the revised submission, we will deposit all code used to program both the BlueHub and BlueBerry devices, together with the Gerber files required for PCB fabrication, alongside the paper.

Reviewer #3 (Public review):

Summary:

This study presents a novel device for wireless control of optogenetic stimulation of the mouse brain, the Blueberry, using Bluetooth Low Energy (BLE) communication for parallel activation of up to 4 devices through an Arduino interface. The authors also present two types of brain implants for light delivery that can be connected to the Blueberry: one using uLEDs for surface cortical stimulation, and another using optical fibers for intra- or sub-cortical implants. The architecture of the system, including electronics, communication, and programming, is thoroughly described. Because the system was especially designed to be integrated with existing software used for neuroscience behavioral experiment for closed-loop experiments, validation of the system is shown on two different scenarios: a learning task in a "infinite" Y-maze, where light delivery at precise locations conditions arm choice for navigation; and a social interaction analysis where 3 animals are simultaneously stimulated in order to alter social dynamics among the group.

Strengths:

(1) The full system can be built by individual labs with simple PCB printing, off-the-shelf components, and readily available hardware (Arduino) for widespread dissemination.

(2) Four headstages can be controlled in parallel for simultaneous experiments with multiple mice.

(3) Validation across different relevant behavioral tests, demonstrating the potential of integrating Bluberry in closed-loop setups.

We thank the reviewer for the statement.

Weaknesses:

(1) Some details in the manuscript regarding system characterization (latency, battery life, etc) are included only in the supplementary materials.

As correctly mentioned, in the revised manuscript we will move the necessary quantifications from supplementary section to main section.

(2) The practical details of integration with other commercial and open-source software used for the closed-loop experiments, which could help third-party researchers interested in using the system, are lacking sufficient detail.

We will clarify this point more clearly in the revised manuscript.

(3) System range (3 meters reported) is limited for a BLE device.

The system range reported is the range considered as reliable communication range. In the revised manuscript we quantify this problem by reporting the Received Signal Strength (RSS) value for multiple BlueBerry devices across varying distances.  

(4) Light output amplitude is not programmable, limiting the choice of stimulation protocols and LEDs used.

That is indeed a limitation of our system, we will investigate the feasibility of integrating programmable stimulation protocols in the updated version of BlueBerry device.

(5) Thermal modeling of the cortical surface stimulator was not performed, and it is unclear if the brain implant for this purpose is within the safety limits.

We thank the reviewer for this comment. In the revised manuscript, we will clarify that the thermal measurements reported here apply only to the specific superficial implant geometry and stimulation conditions used in this study. Because tissue heating depends strongly on implant design and on parameters such as optical power, pulse width, and stimulation frequency, a general safety statement cannot be made for all possible implant configurations. Since the primary goal of this work is to present the wireless device platform rather than to validate a particular implant design, thermal safety should be evaluated individually for each implant and stimulation paradigm.

(6) The paper is missing a comparison with other state-of-the-art devices for wireless control of optogenetic stimulation in mice.

In the revised manuscript, we will include a comparison table summarizing our system alongside currently available wireless optogenetic devices.