In this study, Wilczkowski and colleagues investigated the impact of RMTg-VTA and LHb-RMTg stimulation on cocaine-seeking behavior using optogenetics. The goal was dissect the causal role of these inhibitory nuclei on drug-seeking behavior, and whether stimulation spared CPA and locomotor depression. Stimulation of both RMTg-VTA and LHb-RMTg robustly attenuated lever pressing for cocaine-paired cues, but did not affect inactive lever presses. The LHb-RMTg stimulation resulted in reduced active lever presses even 24 hrs later. Conversely, stimulation of these projections did not significantly induce CPA/CPP, reduce locomotor activity, or affect time spent in the center of the open field. Supplementary results show stimulation of these pathways also effectively reduced VTA DA cell firing. The study is well-designed and executed, and the authors do a good job of addressing the limitations of the study. There are a few concerns to be addressed:

Major:
1) Although statistical results show no effect on CPA/CPP at post-test or locomotor activity, there does seem to be some mild effect of stimulation in opposite directions for the RMTg and LHb. The LHb figure shows a somewhat moderate effect during the direct stimulation sessions where the animals show less time on the laser paired side. Perhaps a bit more clear is the effect on locomotor activity, where LHb stimulation did in fact reduce (p=0.05) locomotor activity. The lack of significance could be due to power (which the authors address) but additional statistical tests might be able to address the current data. Perhaps a repeated measures ANOVA looking at the effects on CPA/CPP including all groups in the model (conditioning sessionviruscircuit) may reveal a significant interaction for CPA that would be informative. Furthermore, including locomotor activity and CPA behavior during these stimulation sessions as a covariate for the cocaine seeking would help to evaluate the role of potential aversion effects.

2) It is unclear if the CPA/CPP animals are distinct from the cocaine-seeking animals. If they are the same animals please indicate when these sessions were run.

3) The discussion goes into a lot of what these circuits do generally that is a little beyond the scope of the current study. Reducing the discussion to points most relevant for the data is recommended.

Minor:
1) Figure 2 is a little confusing to follow. It is recommended that the authors either present the data in sequence (training followed by seeking for each circuit), or present just the seeking data back to back for each circuit followed by the training data (how it is presented in the text). Also, please add individual data points to the bar graphs for clarity and consistency.

2) Presentation of p values is inconsistent where sometimes specific values are given, others are presented as p<0.05. Please provide specific values, ideally up to 3 digits after the decimal point.

In this revised manuscript Wilczkowski and colleagues included additional analyses and revisions to figures that have improved the interpretability of the results. The results are interesting and relevant to the field of addiction and motivation. No further revisions are recommended.