A contextual fear conditioning paradigm in head-fixed mice exploring virtual reality

Reviewer #1 (Public review):

The authors set out to develop a contextual fear learning (CFC) paradigm in head-fixed mice that would produce freezing as the conditioned response. Typically, lick suppression is the conditioned response in such designs, but this (1) introduces a potential confounding influence of reward learning on neural assessments of aversion learning and (2) does not easily allow comparison of head-fixed studies with extensive previous work in freely moving animals, which use freezing as the primary conditioned response.

The first part of this study is a report on the development and outcomes of 3 variations of the CFC paradigm in a virtual reality environment. The fundamental design is strong, with head-fixed mice required to run down a linear virtual track to obtain a water reward. Once trained, the water reward is no longer necessary and mice will navigate virtual reality environments. There are rigorous performance criteria to ensure that mice that make it to the experimental stage show very low levels of inactivity prior to fear conditioning. These criteria do result in only 40% of the mice making it to the experimental stage, but high rates of activity in the VR environment are crucial for detecting learning-related freezing. It is possible that further adjustments to the procedure could improve attrition rates.

Paradigm versions 1 and 2 vary the familiarity of the control context while paradigm versions 2 and 3 vary the inter-shock interval. Paradigm version 1 is the most promising, showing the greatest increase in conditioned freezing (~40%) and good discrimination between contexts (delta ~15-20%). Paradigm version 2 showed no clear evidence of learning - average freezing at recall day 1 was not different than pre-shock freezing. First-lap freezing showed a difference, but this single-lap effect is not useful for many of the neural circuit questions for which this paradigm is meant to facilitate. Also, the claim that mice extinguished first-lap freezing after 1 day is weak. Extinction is determined here by the loss of context discrimination, but this was not strong to begin with. First-lap freezing does not appear to be different between Recall Day 1 and 2, but this analysis was not done. Paradigm version 3 has some promise, but the magnitude of the context discrimination is modest (~10% difference in freezing). Thus, further optimization of the VR CFC will be needed to achieve robust learning and extinction. This could include factors not thoroughly tested in this study, including context pre-exposure timing and duration and shock intensity and frequency.

The second part of the study is a validation of the head-fixed CFC VR protocol through the demonstration that fear conditioning leads to the remapping of dorsal CA1 place fields, similar to that observed in freely moving subjects. The results support this aim and largely replicate previous findings in freely moving subjects. One difference from previous work of note is that VR CFC led to the remapping of the control environment, not just the conditioning context. The authors present several possible explanations for this lack of specificity to the shock context, further underscoring the need for further refinement of the CFC protocol before it can be widely applied. While this experiment examined place cell remapping after fear conditioning, it did not attempt to link neural activity to the learned association or freezing behavior.

In summary, this is an important study that sets the initial parameters and neuronal validation needed to establish a head-fixed CFC paradigm that produces freezing behaviors. In the discussion, the authors note the limitations of this study, suggest the next steps in refinement, and point to several future directions using this protocol to significantly advance our understanding of the neural circuits of threat-related learning and behavior.

Reviewer #2 (Public review):

Summary:

In this manuscript, Krishnan et al devised three paradigms to perform contextual fear conditioning in head-fixed mice. Each of the paradigms relied on head-fixed mice running on a treadmill through virtual reality arenas. The authors tested the validity of three versions of the paradigms by using various parameters. As described below, I think there are several issues with the way the paradigms are designed and how the data are interpreted. Moreover, as Paradigm 3 was published previously in a study by the same group, it is unclear to me what this manuscript offers beyond the validations of parameters used for the previous publication. Below, I list my concerns point-by-point, which I believe need to be addressed to strengthen the manuscript.

Major comments

(1) In the analysis using the LME model (Tables 1 and 2), I am left wondering why the mice had increased freezing across recall days as well as increased generalization (increased freezing to the familiar context, where shock was never delivered). Would the authors expect freezing to decrease across recall days, since repeated exposure to the shock context should drive some extinction? This is complicated by the analysis showing that freeing was increased only on retrieval day 1 when analyzing data from the first lap only. Since reward (e.g., motivation to run) is removed during the conditioning and retrieval tests, I wonder if what the authors are observing is related to decreased motivation to perform the task (mice will just sit, immobile, not necessarily freezing per se). I think that these aspects need to be teased out.

(2) Related to point 1, the authors actually point out that these changes could be due to the loss of the water reward. So, in line 304, is it appropriate to call this freezing? I think it will be very important for the authors to exactly define and delineate what they consider as freezing in this task, versus mice just simply sitting around, immobile, and taking a break from performing the task when they realize there is no reward at the end.

(3) In the second paradigm, mice are exposed to both novel and (at the time before conditioning) neutral environments just before fear conditioning. There is a big chance that the mice are 'linking' the memories (Cai et al 2016) of the two contexts such that there is no difference in freezing in the shock context compared to the neutral context, which is what the authors observe (Lines 333-335). The experiment should be repeated such that exposure to the contexts does not occur on the conditioning day.

(4) On lines 360-361, the authors conclude that extinction happens rapidly, within the first lap of the VR trial. To my understanding, that would mean that extinction would happen within the first 5-10 seconds of the test (according to Figure S1E). That seems far too fast for extinction to occur, as this never occurs in freely behaving mice this quickly.

(5) Throughout the different paradigms, the authors are using different shock intensities. This can lead to differences in fear memory encoding as well as in levels of fear memory generalization. I don't think that comparisons can be made across the different paradigms as too many variables (including shock intensity - 0.5/0.6mA can be very different from 1.0 mA) are different. How can the authors pinpoint which works best? Indeed, they find Paradigm 3 'works' better than Paradigm 2 because mice discriminate better between the neutral and shock contexts. This can definitely be driven by decreased generalization from using a 0.6mA shock in Paradigm 3 compared to 1.0 mA shock in Paradigm 2.

(6) There are some differences in the calcium imaging dataset compared to other studies, and the authors should perform additional testing to determine why. This will be integral to validating their head-fixed paradigm(s) and showing they are useful for modeling circuit dynamics/behaviors observed in freely behaving mice. Moreover, the sample size (number of mice) seems low.

(7) It appears that the authors have already published a paper using Paradigm 3 (Ratigan et al 2023). If they already found a paradigm that is published and works, it is unclear to me what the current manuscript offers beyond that initial manuscript.

(8) As written, the manuscript is really difficult to follow with the averages and standard error reported throughout the text. This reporting in the text occurred heterogeneously throughout the text, as sometimes it was reported and other times it was not. Cleaning this reporting up throughout the paper would greatly improve the flow of the text and qualitative description of the results.

Reviewer #3 (Public review):

Summary:

Krishnan et al. present a novel contextual fear conditioning (CFC) paradigm using a virtual reality (VR) apparatus to evaluate whether conditioned context-induced freezing can be elicited in head-fixed mice. By combining this approach with two-photon imaging, the authors aim to provide high-resolution insights into the neural mechanisms underlying learning, memory, and fear. Their experiments demonstrate that head-fixed mice can discriminate between threat and non-threat contexts, exhibit fear-related behavior in VR, and show context-dependent variability during extinction. Supplemental analyses further explore alternative behaviors and the influence of experimental parameters, while hippocampal neuron remapping is tracked throughout the experiments, showcasing the paradigm's potential for studying memory formation and extinction processes.

Strengths:

Methodological Innovation: The integration of a VR-based CFC paradigm with real-time two-photon imaging offers a powerful, high-resolution tool for investigating the neural circuits underlying fear, learning, and memory.

Versatility and Utility: The paradigm provides a controlled and reproducible environment for studying contextual fear learning, addressing challenges associated with freely moving paradigms.

Potential for Broader Applications: By demonstrating hippocampal neuron remapping during fear learning and extinction, the study highlights the paradigm's utility for exploring memory dynamics, providing a strong foundation for future studies in behavioral neuroscience.

Comprehensive Data Presentation: The inclusion of supplemental figures and behavioral analyses (e.g., licking behaviors and variability in extinction) strengthens the manuscript by addressing additional dimensions of the experimental outcomes.

Weaknesses:

Characterization of Freezing Behavior: The evidence supporting freezing behavior as the primary defensive response in VR is unclear. Supplementary videos suggest the observed behaviors may include avoidance-like actions (e.g., backing away or stopping locomotion) rather than true freezing. Additional physiological measurements, such as EMG or heart rate, are necessary to substantiate the claim that freezing is elicited in the paradigm.

Analysis of Extinction: Extinction dynamics are only analyzed through between-group comparisons within each Recall day, without addressing within-group changes in behavior across days. Statistical comparisons within groups would provide a more robust demonstration of extinction processes.

Low Sample Sizes: Paradigm 1 includes conditions with very low sample sizes (N=1-3), limiting the reliability of statistical comparisons regarding the effects of shock number and intensity. Increasing sample sizes or excluding data from mice that do not match the conditions used in Paradigms 2 and 3 would improve the rigor of the analysis.

Potential Confound of Water Reward: The authors critique the use of reward in conjunction with fear conditioning in prior studies but do not fully address the potential confound introduced by using water reward during the training phase in their own paradigm.