Neuroelectrophysiology-Compatible Electrolytic Lesioning

Reviewer #1 (Public Review):

In the paper, the authors illustrated a novel method for Electrolytic Lesioning through a microelectronics array. This novel lesioning technique is able to perform long-term micro-scale local lesions with a fine spatial resolution (mm). In addition, it allows a direct comparison of population neural activity patterns before and after the lesions using electrophysiology. This new technique addresses a recent challenge in the field and provides a precious opportunity to study the natural reorganization/recovery at the neuronal population level after long-term lesions. It will help discover new causal insights investigating the neural circuits controlling behavior.

Several minor concerns are summarized below:

It was not always clear what the lesion size was. This information is important for future applications, for example, in the visual cortex, where neurons are organized in retinotopy patterns.

It would be helpful if the author could add some discussion about whether and how this method could be used in other types of array/multi-contact electrodes, such as passive neuropixels, S-probes, and so on. In addition, though an op-amp was used in the design, it would still be helpful if the author could provide a recommended range for the impedance of the electrodes.

Reviewer #2 (Public Review):

This work by Bray et al. presented a customized way to induce small electrolytic lesions in the brain using chronically implanted intracortical multielectrode arrays. This type of lesioning technique has the benefit of high spatial precision and low surgical complexity while allowing simultaneous electrophysiology recording before, during, and after the lesion induction. The authors have validated this lesioning method with a Utah array, both ex vivo and in vivo using pig models and awake-behaving rhesus macaques. Given its precision in controlling the lesion size, location, and compatibility with multiple animal models and cortical areas, the authors believe this method can be used to study cortical circuits in the presence of targeted neuronal inactivation or injury and to establish causal relationships before behavior and cortical activity.

Strengths:

Great presentation of design considerations that addressed the gaps of current lesioning and neuronal inactivation methods, especially the cross-compatibility that allows this method to be used across different cortical regions and in different animal models, making it easy to be adopted into a variety of electrophysiology studies.

This method can induce lesions that are highly precise and repeatable in size and location, allowing for robust investigation of neuronal circuit function. When combined with the ability to record without disruption both in the acute and chronic phase after lesioning, this would create a great tool to study neural adaptation and reorganization.

The customized current source is simple, low-cost, yet effective in delivering precise, controllable current for electrolytic lesioning, and thus easy to adopt for a range of neuroscience applications.

Extensive ex vivo testing and validation were performed before moving into in vivo and eventually nonhuman primate (NHP) experiments, successfully reducing animal use.

Weaknesses:

In many of the figures, it is not clear what is shown and the analysis techniques are not well described.

The flexibility of lesioning/termination location is limited to the implantation site of the multielectrode array, and thus less flexible compared to some of the other termination methods outlined in Appendix 2.

Although the extent of the damage created through the Utah array will vary based on anatomical structures, it is unclear what is the range of lesion volumes that can be created with this method, given a parameter set. It was also mentioned that they performed a non-exhaustive parameter search for the applied current amplitude and duration (Table S1/S2) to generate the most suitable lesion size but did not present the resulting lesion sizes from these parameter sets listed. Moreover, there's a lack of histological data suggesting that the lesion size is precise and repeatable given the same current duration/amplitude, at the same location.

It is unclear what type of behavioral deficits can result from an electrolytic lesion this size and type (~3 mm in diameter) in rhesus macaques, as the extent of the neuronal loss within the damaged parenchyma can be different from past lesioning studies.

The lesioning procedure was performed in Monkey F while sedated, but no data was presented for Monkey F in terms of lesioning parameters, lesion size, recorded electrophysiology, histological, or behavioral outcomes. It is also unclear if Monkey F was in a terminal study.

As an inactivation method, the electrophysiology recording in Figure 5 only showed a change in pairwise comparisons of clustered action potential waveforms at each electrode (%match) but not a direct measure of neuronal pre and post-lesioning. More evidence is needed to suggest robust neuronal inactivation or termination in rhesus macaques after electrolytic lesioning. Some examples of this can be showing the number of spike clusters identified each day, as well as analyzing local field potential and multi-unit activity.

The advantages over recently developed lesioning techniques are not clear and are not discussed.

There is a lack of quantitative histological analysis of the change in neuronal morphology and loss.

There is a lack of histology data across animals and on the reliability of their lesioning techniques across animals and experiments.

There is a lack of data on changes in cortical layers and structures across the lesioning and non-lesioning electrodes.

Author Response:

We thank the reviewers and editor for their feedback, which we will carefully consider as we revise the manuscript. We aim to provide more detail on how this technique could be used with other probes, ideally showing experimental data to support this use. We will add further detail of the histology from our ex vivo ovine and porcine and in vivo porcine testing. We will also provide a more thorough comparison of our technique to other recently developed lesioning techniques. In order to provide more complete evidence that our technique perturbs local neuron populations, we will refine the action potential analysis presented before and after lesions in non-human primates. In addition to providing further clarity of the method, we will include more non-human primate data where possible.