Zona incerta distributes a broad movement signal that modulates behavior

Reviewer #1 (Public Review):

This study by Hormigo et al. examines the relationship between activity in the zona incerta (ZI) and behavior. The authors aim to assess the hypothesis that the ZI might mediate a general behavioral function, namely the distribution of information about ongoing movement to other brain areas that regulate behavior. Given the heterogeneity of prior literature on the ZI, this topic is important and interesting. The study employs a strong diversity of technical approaches, spanning electrophysiological recordings, calcium imaging, optogenetics, virally-mediated cell-type ablation, and several behavioral assays. The output is a large dataset where each experiment is useful and interesting, and together, the results could be interpreted as consistent with the prospect of the ZI mediating a general function. However, there are notable weaknesses in the current version of this paper. First, it is unclear whether the experiments and analyses were set up to be able to rule out more specific candidate functions of the ZI. Second, many important details of the experiments and their results are hard to decipher given the current descriptions and presentations of the data.

The paper could be significantly strengthened by including more details from each experiment, stronger justifications for the limited behaviors and experimental analyses performed, and, finally, a broader analysis of how the recorded activity in the ZI relates to behavioral parameters.

(1) Anatomical specification: The ZI contains many distinct subdivisions--each with its own topographically organized inputs/outputs and putative functions. The current manuscript doesn't reference these known divisions or their behavioral distinctions, and one cannot tell exactly which portion(s) of the ZI was included in the current study.

Moreover, the elongated structure of the ZI makes it very difficult to specifically or completely infect virally. The data could be better interpreted if the paper included basic information on the locations of recordings, the extent of the AAV spread in the ZI in each viral experiment, and what fraction of infected neurons were inside versus outside ZI.

(2) Electrophysiological recording on the treadmill: The authors are commended for this technically very difficult experiment. The authors do not specify, however, how they knew when they were recording in ZI rather than surrounding structures, particularly given that recording site lesions were only performed during the last recording session. A map of the locations of the different classes of units would be valuable data to relate to the literature.

(3) The rationale of the analysis of activity with respect to "movement peak": It is unclear why the authors did not assess how ZI activity correlates with a broad set of movement parameters, but rather grouped heterogeneous behavioral epochs to analyze firing with respect to "movement peaks".

(4) The display of mean categorical data in various figures is interesting, however, the reader cannot gather a very detailed view of ZI firing responses or potential heterogeneity with so little information about their distributions.

(5) Somatosensory firing responses in ZI: It is unclear why the authors chose the specific stimuli used in the study. How often did they evoke reflexive motor responses? What was the latency of sensory-evoked responses in ZI activity and the latency of the reflexive movement?

(6) It would be valuable to see example traces in Figure 3 to get a better sense of the time course and contexts under which Ca signals in ZI tracks movement. What is the typical latency? What is the typical range of magnitudes of responses? Does the Ca signal track both fast and slow movements? How are the authors sure that there are no movement artifacts contributing to the calcium imaging? It seems there is more information in the dataset that could be valuable.

(7) Figure 4: The rationale for quantifying the F/Fo responses over a 6-second window, rather than with respect to discrete movement parameters, is not well explained. What types of movement are binned in this approach and might this broad binning hinder the ability to detect more specific relationships between activity and movement?

(8) Separation of sensory and motor responses in Figure 5: The current data do not adequately differentiate whether the responses are sensory or motor given the high correlation of the sensory inputs driving motor responses. Because isoflurane can diminish auditory responses early in the auditory pathway, this reviewer is not convinced the isoflurane experiments are interpretable.

(9) Given the broad duration of the mean avoidance response (Fig. 6 C, bottom), it would be useful to know to what extent this plot reflects a prolonged behavior or is the result of averaging different animals/trials with different latencies. Given that the shapes of the F/Fo responses in ZI appear similar across avoids and escapes (Fig. 6D), despite their apparent different speeds and movement durations (Fig 6C), it would be valuable to know how the timing of the F/Fo relates to movement on a trial-by-trial basis.

(10) Lesion quantification: One cannot tell what rostral-caudal extent of ZI was lesioned and quantified in this experiment. It would be easier to interpret if also plotted for each animal, so the reader can tell how reliable the method is. The mean ablation would be better shown as a normalized fraction of cells. Although the authors claim the lesions have little impact on behavior, it appears the incompleteness of the lesions could warrant a more conservative interpretation.

(11) Optogenetics: the location of infected neurons is poorly described, including the rostral-caudal extent and the fraction of neurons inside and outside of ZI. Moreover, it is unclear how strongly the optogenetic manipulations in this study are expected to affect neuronal activity in ZI.

Reviewer #2 (Public Review):

The manuscript presents compelling evidence for the role of the zona incerta area of the brain in regulating movement and sensory stimuli in mice. The study uses an appropriate and validated methodology in line with the current state-of-the-art, including optogenetic manipulation and recording of single-unit activity. The authors' claims and conclusions are well-supported by their data, which includes a comprehensive review of previous research on the zona incerta. Overall, the manuscript provides solid evidence for the role of the zona incerta in regulating movement and sensory processing.

Major strengths and weaknesses of the methods and results.
The zona incerta has many integrative functions that link sensory stimuli with motor responses to guide behavior.
The study explored the activation of zona incerta GABAergic neurons during cued avoidance tasks and found that these neurons activate during goal-directed avoidance movement. Optogenetic manipulation of these neurons affected movement speed and performance during active avoidance tasks.
The findings suggest that the zona incerta area of the brain plays a significant role in regulating movement and responding to salient auditory tones in association with movement in mice. The evidence presented is fundamental and provides a comprehensive review of previous research on the zona incerta and its involvement in various behaviors and sensory processing.

The article is very well written, with a correct hypothesis and a cutting-edge methodology to achieve the expected objectives. Moreover, they use statistical rigorous approaches in the analysis of the results. Also, analyzes are performed using scripts that automate all aspects of data analysis, ensuring their objectivity. The results are very novel, and provide solid evidence for the role of the zona incerta in regulating movement and sensory processing.