An approach for long-term, multi-probe Neuropixels recordings in unrestrained rats

  1. Thomas Zhihao Luo
  2. Adrian Gopnik Bondy
  3. Diksha Gupta
  4. Verity Alexander Elliott
  5. Charles D Kopec
  6. Carlos D Brody

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Abstract

The use of Neuropixels probes for chronic neural recordings is in its infancy and initial studies leave questions about long-term stability and probe reusability unaddressed. Here, we demonstrate a new approach for chronic Neuropixels recordings over a period of months in freely moving rats. Our approach allows multiple probes per rat and multiple cycles of probe reuse. We found that hundreds of units could be recorded for multiple months, but that yields depended systematically on anatomical position. Explanted probes displayed a small increase in noise compared to unimplanted probes, but this was insufficient to impair future single-unit recordings. We conclude that cost-effective, multi-region, and multi-probe Neuropixels recordings can be carried out with high yields over multiple months in rats or other similarly sized animals. Our methods and observations may facilitate the standardization of chronic recording from Neuropixels probes in freely moving animals.

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  1. Version published to 10.7554/elife.59716 on eLife
  2. eLife

    ###Reviewer #3:

    This study provides very informative trends regarding long-term (~4-month) recording with Neuropixels probes in chronically implanted, freely moving rats. This is accomplished by recording across many animals (n = 18) and many recording locations and analyzing the number of single (and multi) units that can be automatically isolated as a function of time since implant, recording location, and other features (e.g. shank orientation). The authors perform these experiments with a modular system that allows the implanting of multiple probes simultaneously in a single rat (here they mostly implanted 1 probe, sometimes 2, once 3) and that allows the removal of probes for re-use in another animal, both of which are also valuable contributions. The analysis of neuron yield is framed in terms of a sum of 2 decaying exponentials model (an initial fast decay of one subpopulation of neurons followed by a slower decay of the remainder) that the authors fit to find the primary features determining neuron yield. The major trends they report include: substantially better yield over time in regions anterior to the bregma and ventral to the most dorsal 2mm of the brain surface. They also show that re-used probes perform essentially as well as new probes in terms of noise and unit quality (e.g. average unit amplitude), and also neuron yield (at least for medial frontal cortex, but see below).

    Major comments:

    The results averaged over many animals and the model are good for extracting the major trends, but there are hints of significant and important variability across animals or probes. Points 1-3 are about this variability, potential sources of variability, and displaying the variability whether or not potential causes can be found.

    1. The results shown in Figure 2, especially the averages in Figure 2H,K, indicate severe losses in unit yield over time for probes implanted posterior of bregma and electrode sites in the dorsal 2mm of the rat brain. However, Figure 2B shows at least one animal (open circles) for which high neuron yield was obtained in motor cortex and dorsal striatum for at least 4 months. First, is this from 1 or 2 probes? Whether it is from 1 or 2 probes, the stability of the recording over time from day 1 is much greater than the other animals, and much better than what is expected from Figure 2H. Is the preservation of units over time for this animal due to the stability of units in dorsal striatum (presumably mostly >2mm below the surface) or also motor cortex?

    2. There are some additional potential causes that could also account for yield differences, and one of these is age of the animal at the time of implant. The authors should list age at implant in their table in Figure 4-supplement 1. The authors should display yield over time as a function of age at implant, and also try adding age as one of the regressors for their model of neuron yield.

    3. Another potential cause is whether the probe is new or reused. The authors showed that probe re-use did not result in statistically different yield for the medial prefrontal cortex. But is this also true for the other brain regions? Does the data in Figure 2 include implants of both new and re-used probes, or only new probes? The authors should try to add whether the probe was new or re-used as a regressor in their neuron yield model.

    Regarding points 1-3, whether or not it is possible to add age or probe newness as regressors in the model, the authors should create a supplementary figure that shows the single unit yield curves as in Figure 2A-C for all probes in all animals: one panel per major brain region (e.g. splitting motor cortex from dorsal striatum from ventral striatum), with one curve per probe. There should be a legend for each panel that gives the (AP,ML,DV) coordinates of the approximate midpoint of the probe's location within that brain region. The legend should also indicate for each probe/curve: the animal, age at time of implant, probe newness, probe tip depth, estimated number of electrodes recorded from in that region, and shank orientation. This will repeat some pieces of information that's in the tables, however it's very useful to see all this information together in a form that would be very valuable for readers, especially experimenters who may want to record from some of the more posterior and dorsal areas. The information that could be gleaned would include knowledge of the variance in yield over time across implant attempts, so they could see if, say, 1 of 3 attempts to implant in a given area may give very good long-term yield.

    1. It is stated starting on Line 172 that "The relative number of units corresponding to the fast- and slowly-decaying subpopulations did not significantly vary across brain regions along either anatomical axis, nor did the rate of decay of the fast population (Figure 2--supplement 3). This suggests that the rapid decline in yield observed in the days after surgery may be due to a process that is relatively uniform across brain regions."

    The support for this statement can be seen in the indicated Figure 2-supplement 3. On the other hand, the point is made (and shown in Figure 2-supplement 4) that there is no loss of units in mPFC over time. This is apparently at odds with the Line 172 statement and model assumption of a fixed fraction of fast-decaying units. Was a model tried in which alpha varies with location? If the Line 172 statement is ultimately kept, there should at least be a comment made there that the most anterior, ventral regions appear to differ from the model's assumption/interpretation.

  3. eLife

    ###Reviewer #2:

    In this paper, the authors report a device that can be used to implant and later explant Neuropixels probes in freely moving rats. The device consists of an adaptor, an internal holder and an external chassis. The chassis protects the probe, is attached to the animal's head via adhesive cement and acrylic. The internal part can be explanted at the end of the experiment, allowing the NP probe to be re-used.

    The work builds on existing technology in important ways: the authors examined the long-term yield across different brain regions, they more extensively assessed the feasibility of probe reuse compared to previous work, and they evaluated probe performance over a long period of time and also after explanation (measuring the input referred noise of explanted probes in saline). It was also impressive that they used a cohort of 18 rats to evaluate performance of both the animals and the probe, and that they were able to implant up to 3 NP probes at a time. Because of the importance of using freely moving animals in Neuroscience research, and the differences between rats and mice that necessitate modifications on existing technology, this paper is timely and likely to be very useful to a sizeable group of researchers. My suggestions are aimed at furthering the usefulness of this "Tools and Resources" paper for investigators who wish to use this important technology.

    At the moment, the majority of the paper seems aimed at evaluating the performance of the device as a function of time, depth and location. This performance evaluation was useful, very carefully done, and makes important points that aid in the interpretation of other papers (such as the unusual stability of recordings in mPFC reported in previous papers). Nonetheless, readers are likely interested in the paper because they wish to make and implant the device in order to benefit from the scholarly analysis done here. The manuscript does contain very helpful technical details, but these are hard to find and are not front-and-center in the main text. For instance, the material from the "Neuropixels implant procedure" is really helpful and would be critical for anyone who wants to use this technique. But at the moment, that information is in a google doc linked from the associated GitHub, a long way from the main manuscript. This information should be in the main manuscript, either in Results or Methods. Also use of consistent nomenclature across documents would help a lot. I believe the part referred to as the "chassis" in the main text is referred to as the "external" on the google doc with the instructions. Similarly, the part referred to as the "internal" in the google doc is called an "internal holder" in the manuscript.

    A reader hoping to use the device might also benefit from more information on the grounding procedure. The text in the "Implantation" section of the methods was helpful, but more information would be useful, such as where on the probe the ground wire should be connected and how one should fix the grounding wire (tapping the wire and covering with Metabond?). Also, it would be nice to know how one should protect the grounding wire from being touched by the animal. Figure 6 in the google doc protocol is really helpful and should definitely be in the main manuscript. An additional figure showing how to connect the wire to the ground during the surgery would be quite useful. Finally, are the craniotomy and durotomy necessary for grounding? Could one simply connect the grounding wire to a couple of screws on the skull?

  4. eLife

    ###Reviewer #1:

    This manuscript presents new techniques for obtaining chronic recordings using multiple neuropixel probes in rats. The resources, I imagine, will be of high value to the neuroscience community at large. They also address short and long terms unit stability, probe recovery and impact of the probe on behavior. I have only a few minor comments.

    I understand the authors rationale to avoid manual curation but there have been reports of inconsistencies in the identification of units across different sorters. Did the authors consider comparing their kilosort unit identification with manual curation or another sorting software?

    The authors speculate in the discussion about the possible reason for the slow loss of units. It wasn't quite clear to me however, what types of changes might improve this loss?

    Figure 2 is perhaps one of the most informative findings but I wonder how applicable this will be to future probe iterations. Do the authors have a hypothesis for what features of the probe might contribute (or not contribute) to the long term loss of units?

  5. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 2 of the manuscript. Lisa Giocomo (Stanford University School of Medicine) served as the Reviewing Editor.

    ###Summary:

    This manuscript presents new techniques for obtaining chronic recordings using multiple neuropixel probes in rats. The study provides very informative trends regarding long-term (~4-month) recording with Neuropixels probes in chronically implanted, freely moving rats. This is accomplished by recording across many animals (n = 18) and many recording locations and analyzing the number of single (and multi) units that can be automatically isolated as a function of time since implant, recording location, and other features (e.g. shank orientation). The authors perform these experiments with a modular system that allows the implanting of multiple probes simultaneously in a single rat (here they mostly implanted 1 probe, sometimes 2, once 3) and that allows the removal of probes for re-use in another animal, both of which are also valuable contributions. The work builds on existing technology in important ways: the authors examined the long-term yield across different brain regions, they more extensively assessed the feasibility of probe reuse compared to previous work, and they evaluated probe performance over a long period of time and also after explanation (measuring the input referred noise of explanted probes in saline). Because of the importance of using freely moving animals in Neuroscience research, and the differences between rats and mice that necessitate modifications on existing technology, this paper is timely and likely to be very useful to a sizable group of researchers.

  6. Version published to 10.1101/2020.04.13.039305 on bioRxiv