The open field assay is influenced by room temperature and by drugs that affect core body temperature

Background The open field assay is used to study anxiety-related traits and anxiolytic drugs in rodents. This assay entails measuring locomotor activity and time spent in the center of a chamber that is maintained at ambient room temperature. However, the ambient temperature in most laboratories varies daily and seasonally and can differ between buildings. We sought to evaluate how varying ambient temperature and core body temperature (CBT) affected open field locomotor activity and center time of male wild-type (WT, C57BL/6) and Transient Receptor Potential Subfamily M Member 8 ( Trpm8) knock-out ( Trpm8 -/- ) mice. TRPM8 is an ion channel that detects cool temperatures and is activated by icilin. Methods Mice were placed in the open field at 4°C and 23°C for 1 hour. Distance traveled and time spent in the center were measured. Mice were injected with icilin, M8-B, diazepam, or saline, and changes in activity level were recorded. Results The cooling agent icilin increased CBT and profoundly reduced distance traveled and center time of WT mice relative to controls. Likewise, cooling the ambient temperature to 4°C reduced distance traveled and center time of WT mice relative to Trpm8 -/- mice. Conversely, the TRPM8 antagonist (M8-B) reduced CBT and increased distance traveled and center time of WT mice when tested at 4°C. The TRPM8 antagonist (M8-B) had no effect on CBT or open field behavior of Trpm8 -/- mice. The anxiolytic diazepam reduced CBT in WT and Trpm8 -/- mice. When tested at 4°C, diazepam increased distance traveled and center time in WT mice but did not alter open field behavior of Trpm8 -/- mice. Conclusions Environmental temperature and drugs that affect CBT can influence locomotor behavior and center time in the open field assay, highlighting temperature (ambient and core) as sources of environmental and physiologic variability in this commonly used behavioral assay.


Introduction
Drugs used to treat neurological and neuropsychiatric disorders are typically evaluated in rodent models for safety and efficacy prior to use in humans. 1 Characterizing animal models of neuropsychiatric disorders often relies on behavioral traits such as motor function, social interactions, anxiety-like and depressive-like behavior, substance dependence, and various forms of cognitive function. 2Due to the complexity of most behavior tests, researchers must carefully consider the sources of variability introduced by experimenters, testing environments, and intraspecies differences.
Rodent physiology and behavior are influenced by environmental temperature. 3,4For example, an innocuous cold stimulation at 15°C altered sleeping, rearing, climbing, and eating behavior in wild-type (WT) mice. 5This cold stimulation did not alter these behaviors in mutant mice lacking the Transient Receptor Potential Subfamily M Member 8 (TRPM8) cation channel, which is a receptor for menthol and icilin (mint-derived and synthetic cooling compounds, respectively) and plays an important role in thermosensation. 5,6Additionally, mice deficient in uncoupling protein 1 (UCP-1), a key metabolic regulator highly expressed in brown adipose tissue, were reported to display selective enhancement of anxiety-related behavior exclusively under thermogenic conditions (23°C), but not at thermoneutrality (29°C). 7vironmental temperature sensation and perception is also influenced by core body temperature (CBT).Alterations to CBT can be a consequence of physiological changes associated with disease state, exercise, metabolic function, and hormonal changes.][10][11] It is currently unclear if drugs such as these, which are used to treat neurological and neuropsychiatric disorders, affect CBT directly or indirectly, and if the behavioral tasks that are commonly used to study these drugs are influenced by changes in CBT.
Here we sought to evaluate how ambient temperature and changes in CBT influence locomotor activity and center time in the open field assay-an assay that is commonly used to study anxiolytic drugs and animal models of anxiety.In addition to using WT mice, we also used Trpm8 -/-mice, which lack the primary receptor for cool temperature sensation in mammals, as well as drugs that activate (icilin) or antagonize (M8-B) this receptor.Diazepam was also evaluated as a model anxiolytic drug.By precisely controlling environmental temperature and TRPM8 activity (genetically and pharmacologically), we found that commonly used measures associated with the open field assay are profoundly sensitive to ambient temperature and CBT.To enhance rigor and reproducibility, we recommend that ambient and core temperature be precisely controlled when performing the open field assay.Moreover, drugs that increase activity and center time in the open field test may do so via thermoregulatory mechanisms, independent of effects on anxiety.

Mice
Animal protocols in this study were approved by the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill and were performed in accordance with these guidelines and regulations at the University of North Carolina at Chapel Hill (NIH/PHS Animal Welfare Assurance Number D16-00256 A3410-01, expiration April 30, 2025; USDA Animal Research Facility Registration Number 55-R-0004; AAALAC Institutional Number #329, re-accreditation November 2020).All data presented in this study are from male mice obtained from crossing Trpm8 +/- male with Trpm8 +/-female mice.Trpm8 mutant mice were obtained from Jackson Laboratories (B6.129P2-Trpm8 tm1Jul / J; stock #008198).Mice were raised in a facility with a 12 h:12 h light:dark cycle with ad libitum access to food (Teklad 2020X, Envigo, Huntingdon, UK) and water.All mice were tested at 8-12 weeks of age.Mice were excluded if they showed signs of distress or lethargy.Genomic DNA was isolated from tail clips using Proteinase K. Genotyping was

REVISED Amendments from Version 3
To address the Reviewers comments, the Abstract Methods were corrected to state an open-field duration of 1 hour.The use of male mice only was re-emphasized and stated as a limitation in the discussion.The rationale for doses tested was elaborated as part of the previous revision and an additional reference was included.It was highlighted that skin temperature was not tested, rather core body temperature was measured with a rectal thermometer.An additional reference was included and described to demonstrate that previous studies have shown a rapid change in core body temperature post-icilin injection using thermal telemetry and at doses lower than the one used in the current study.This is to highlight that our results may be due to the use of a less-sensitive device and lower sample size.Finally, our response emphasizes that the behavior of Diazepam and M8-B injected mice are compared with saline-injected controls in Figure 5.The Reviewer is also guided to a new Figure 3, which was part of the previous revision and better represents the experiment.
Any further responses from the reviewers can be found at the end of the article performed by polymerase chain reaction (PCR) amplification of genomic DNA with primers: WT Forward 5 0 -CCT TGG CTG CTG GAT TCA CAC AGC-3 0 , Mutant Reverse 5 0 -CAG GCT GAG CGA TGA AAT GCT GAT CTG-3 0 , WT Reverse 5 0 -GCT TGC TGG CCC CCA AGG CT-3 0 .Premade buffers along with the platinum Taq were used for amplification (Invitrogen) to amplify DNA in a BioRad DNA Engine (PTC-200).Nucleotides were obtained from Qiagen.Amplification cycle was as follows: 94°C for 3 min, 36 cycles of 94°C for 30 s, 68°C for 60 s and 72°C for 60 s.A final incubation was performed at 72°C for 2 min.The same mice were used for tests in Figures 3-6.All tests were performed 1 week apart.

Body temperature
CBT was assessed using a Digi-Sense Thermocouple Meter (Fisher 13-245-293) to measure rectal temperature.Male mice were acclimated to the procedure 2x each day for one week prior to testing.Temperature was measured 30, 60 and 90 minutes post drug administration for diazepam and M8-B.To assess the effects of icilin on CBT, measures were taken every 15 minutes.

Open-field test
Exploratory activity in a novel environment was assessed by a 1 h trial in an open-field chamber (45 cm Â 45 cm Â 40 cm) 30 minutes post icilin or diazepam administration, and 1 h following M8-B administration.The total distance moved by each mouse in the open arena, and time spent in the center region of the open-field, were recorded by camera (Sony) connected to the EthoVision software (Noldus Wageningen).Testing was performed at room temperature (23°C, lighting: 660 lux) or in the cold room (4°C, lighting: 400 lux).

Data analysis
Data were graphed using GraphPad Prism (v9.5.0) and analyzed with a paired t-test approach.Open field comparing genotype and temperature was analyzed using mixed effect analysis with multiple comparisons, Sidak (Figure 3).All studies were randomized, double blind, and vehicle-controlled consisting of 6-14 mice.All animals were group housed.

Results
Room temperature in a laboratory setting must be well-controlled to eliminate daily and seasonal fluctuations We found that the ambient temperature varied throughout the day (data not shown, can be found as Underlying data 20 ), week, and season in a room that we previously used for behavioral studies (Figure 1A-C, Building 1). 20Temperature fluctuations are presumably common in laboratory settings because building heating, ventilation, and air conditioning are set to maximize human comfort during the work day and minimize energy use during off-peak hours, like evenings and on weekends.Moreover, room temperature was over 4°C warmer in the winter months and 2°C warmer in the summer months in a different laboratory located in a different building (Building 2; Figure 1B and C).Temperature differences over days, seasons, and buildings represent a major source of variability, especially for behavioral experiments that are carried out at "room temperature" and that could be influenced by temperature.To address this source of uncontrolled variability, we worked with the university to custom engineer the heating, ventilation, and air conditioning within our behavioral room so that the temperature could be precisely maintained at a set temperature (we chose 23°C) without fluctuations over the course of the day and seasons (Figure 1B and C).This temperature-controlled room, and a cold room set at 4°C, were used for all subsequent behavioral studies.

TRPM8 agonist impacts CBT and reduces open field behavior in WT mice
TRPM8 is a principal sensor of cold temperatures in mammalian primary sensory neurons. 12,13To explore the impact of TRPM8 stimulation on open field behavior, we administered (i.p.) 50 mg/kg bw icilin, a TRPM8 agonist, or DMSO control to WT mice.Previous studies have shown a rapid change in core body temperature post-icilin injection using thermal telemetry and at doses lower than the one used in the current study, and these significant changes in CBT lasted at least 90 minutes. 14Using a less sensitive device to measure CBT, we did detect an Icilin-induced significant increase in CBT beginning at 90 minutes post injection, consistent with these studies (Figure 2A).We likely did not see an earlier change in CBT due to our low sample size and less sensitive temperature detection method.To assess the impact of increased CBT on open field behavior, we administered 50 mg/kg bw icilin, waited 10 minutes, and then measured distance traveled and time spent in the center (Figure 2B and C).Icilin administration significantly reduced activity in the open field, suggesting that an increase in CBT led to a reduction in open field behavior.
Cooling environment to 4°C reduces open field behavior in WT but not Trpm8 -/-mice Stimulation of TRPM8 channels with icilin impacts the behavior of WT mice in the open field (Figure 2).Thus, we hypothesized that cold stimulation of TRPM8 channels would similarly impact open field behavior and that mice lacking TRPM8 channels would resist the effect of cold stimulation on open field behavior.We found that WT and Trpm8 -/-mice display similar distance traveled and time spent in the center when tested at 23°C (Figure 3A and B).When mice were tested at 4°C, an effect of genotype was revealed, in which the WT mice display reduced distance traveled and center time compared to Trpm8 -/-mice (Figure 3A and B).

M8-B antagonism of TRPM8 channels increases open field behavior in WT mice
We next used a TRPM8 antagonist (M8-B) to block cold-induced stimulation of TRPM8 channels in WT mice.Administration (i.p.) of M8-B at 12 mg/kg bw decreased the CBT of WT but not Trpm8 -/-mice at >1 h post injection at room temperature (Figure 4A).Thus, mice were placed in the open field chamber 1 h following M8-B administration.We found that TRPM8 antagonist administration partially recovered the reduction in open field behavior at 4°C in WT mice (Figure 4B and C, Figure 5A and B).These data suggest that environmental temperature sensation influences open field behavior in mice.
Anxiolytic diazepam reduces CBT in WT and Trpm8 -/-mice Benzodiazepines, such as diazepam, are commonly prescribed to reduce anxiety in humans.Diazepam functions to increase gamma-aminobutyric acid (GABA) in the brain and is used to treat anxiety.To investigate whether the anxiolytic effect of diazepam was associated with a reduction in CBT in mice, WT and Trpm8 -/-mice were administered 2 mg/kg bw of the drug at room temperature.Diazepam exposure led to a reduction in CBT at 30-90 minutes post injection (Figure 6A).This reduction in CBT was associated with a near-significant increase in distance traveled and center time displayed by WT but not Trpm8 -/-mice, when tested at 4°C (Figure 6B and C, Figure 5).

Discussion
In this study we compared the behavioral effects of the anxiolytic drug diazepam, with the effects of other drugs that alter cool temperature sensation and CBT, including icilin and M8-B in male WT and Trpm8 -/-mice.We observed CBT and open field behavioral effects of icilin and M8-B in WT mice, but not Trpm8 -/-mice.
The effect of drugs on core body temperature may be mediated by acting on any component of the thermoregulatory system.These components include heat production, heat conservation, and thermosensing-related pathways within the nervous system that coordinate thermoregulation. 8Clark et al., 8 present a thorough study of drug-induced changes in body temperature and provide a source of information on interactions between certain drugs and the thermoregulatory system.The data present an extensive review of the magnitude of body temperature changes induced by psychoactive compounds while taking into account the species, administration route, dose, and environmental temperature differences.However, with psychoactive drugs, including the drugs used in this study, it is difficult to discern the underlying cause of behavior effects.6][17][18] DMSO at the concentration used to dissolve icilin and M8-B have also been reported to be well tolerated by mice. 19Of note, our vehicle controls were saline injected in Figure 5, which may limit the interpretations of this study.Considering the effects of drug-induced changes on body temperature and the impact of CBT on behavior, studies using rodent models of psychological disorders should consider potential alterations to the perception of environmental temperatures.
The spontaneous behavior of animal models is often used to evaluate the efficacy of drugs used to treat neuropsychiatric disorders.One main concern with animal models is the lack of standardization between laboratories, which can lead to results that are not reproducible.We suggest that stricter testing protocols include assessment of room temperature and control for drug-induced alterations to CBT.For consistency, only male mice were used in this study.However, it should be stated that female mice may display different drug-induced behavioral responses and should be explored in future studies.Ideally, the lighting conditions between all testing environments should be identical.In this study, the animals experienced a slight difference in lighting between the 23°C (660 lux) and 4°C (400 lux) conditions.However, both settings provided very bright conditions and the slight variation is not expected to have altered avoidance behavior.
While the testing environments used in this study (23°C and 4°C) represent a wider temperature range than is observed between laboratories, this proof of principle study demonstrates that temperature can have an influence on behavior.More importantly, in this study we demonstrate that the body temperature changes induced following drug administration could lead to similar physiological responses induced by normal room temperature fluctuations.
By providing greater understanding of the relationship between body temperature and behavior in mice, our data highlight the importance of assessing CBT, environmental temperature, and drug-induced changes to thermoregulation.Thus, consideration of ambient and CBT is a straightforward approach to enhance rigor and reproducibility in studies of neuropsychiatric disorders.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound?Yes

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound?Yes

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate?Yes Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Environmental physiology, Thermoregulation I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
4) The authors are rather measuring the skin temperature and not core body temperature.Can they please explain?They should also discuss how skin temperature correlates with CBT.There are also drugs that can solely influence the skin temperature.It should be noted.

5)
In figure 2, Icilin alter skin temperature 90 mins post-injection.However, the open field starts 10 min after the injection and lasts for 60 mins (i.e.70 mins post-injection).How, then, they concluded that altered temperature influences the open field behavior.Can they please explain? 6) In figure 6, it should be noted that no vehicle-injected mice were used.It is common that skin temperature increases and decreases rapidly upon i.p injections.Without an appropriate control experiments, it is rather difficult to confidently conclude whether diazepam, indeed, reduces body temperature.That is certainly a limitation and should be noted.

7)
Its extremely unclear what is the difference of figure 6 and 3.In this graph it seems that diazepam has no effect at all.The authors should explain and also further discuss these results in conclusion.

Are sufficient details of methods and analysis provided to allow replication by others? Partly
If applicable, is the statistical analysis and its interpretation appropriate?Partly Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Partly
Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Neuroscience
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Author Response 26 Jun 2024

Mark Zylka
In this study, by Jimenez et.al, the effects of an ambient cold temperature (4°C) in the open field are described.In addition, the authors pharmacologically inhibited or activated the cold-sensing TRPM-8 cation receptors, in mice exposed at cold ambient temperature (4°C), and described the interactions between TRPM-8 channel and cold exposure, as observed in the open field test.While is quite an interesting, I would like to encourage the authors to address the following comments: 1) In the main text the open field test lasted for 1 hours.However in the abstract it is mentioned 'Mice were placed in the open field at 4°C and 23°C for 30 minutes'.

Ans:
The open field was run for 1 hour.The abstract method was corrected.
2) Did the authors use male or female mice?And why?It should be always clearly stated.Ans: As stated in the Abstract Background, male WT and TRPM8 knock-out mice were used.This is now restated in the methods and discussion as a limitation."For consistency, only male mice were used in this study.However, it should be stated that female mice may display different drug-induced behavioral responses and should be explored in future studies." 3) Can the authors explain the rationale for the doses tested for all compounds, as well as include references with the effects on these compounds in the open field test?
Ans: This comment was addressed in the previous revision with appropriate reference "Here we selected drug doses that are commonly used and well tolerated by mice in the behavior field."Almeida et al. used 6 mg/kg i.p.M8-B to evaluate the hypothermic action of the drug.We used 12 mg/kg as we didn't see an effect with 6 mg/kg.Vinkers et al. evaluated stress-induced hyperthermia following 1-4 mg/kg diazepam.Padua-Reis et al. also evaluated the anxiolytic effect of 2 mg/kg diazepam.We used 2 mg/kg.New reference: Colburn et al. evaluated icilin-induced jumping and cold sensation following 60 mg/kg icilin administration (new reference).We used 50 mg/kg.
4) The authors are rather measuring the skin temperature and not core body temperature.Can they please explain?They should also discuss how skin temperature correlates with CBT.Ans: There are also drugs that can solely influence the skin temperature.It should be noted.
We did not measure skin temperature.CBT was assessed by measuring rectal temperature in mice, a technique that has been widely used for many years.Limitations of this procedure include distress to the animals, which could increase core temperature.However, however this risk was reduced by acclimating the mice to the procedure 2x each day for one week prior to testing.This was added to the methods.

5)
In figure 2, Icilin alter skin temperature 90 mins post-injection.However, the open field starts 10 min after the injection and lasts for 60 mins (i.e.70 mins post-injection).How, then, they concluded that altered temperature influences the open field behavior.Can they please explain?Ans: Previous studies had already shown a rapid change in core body temperature posticilin injection using thermal telemetry and at doses lower than the one used in the current study, and these significant changes in CBT lasted at least 90 minutes (Knowlton et al, 2011 -New Reference).Using a less sensitive device, we did see an increase in CBT during the open field studies consistent with these studies.We likely did not see a change in CBT earlier due to our low sample size and less sensitive temperature detection method.This information was added to the results section.

6)
In figure 6, it should be noted that no vehicle-injected mice were used.It is common that skin temperature increases and decreases rapidly upon i.p injections.Without an appropriate control experiments, it is rather difficult to confidently conclude whether diazepam, indeed, reduces body temperature.That is certainly a limitation and should be noted.
Ans: To consider temperature changes upon i.p. injections, the results from figures 4 and 6 are compared with their saline-injected vehicle controls in figure 5. Diazepam was dissolved in saline and M8-B was dissolved DMSO.This is included in the methods section.The following limitation is included in the discussion as a previous revision: "Of note, our vehicle controls in Figure 5 were saline injected, which may limit the interpretation of this study."However, "DMSO at the concentration used to dissolve Icilin and M8-B have also been reported to be well tolerated by mice".

7)
Its extremely unclear what is the difference of figure 6 and 3.In this graph it seems that diazepam has no effect at all.The authors should explain and also further discuss these I thank the authors for addressing many of my concerns.Please note that the figure legend for Figure 4A-C is still mislabeled.I believe that the temperature panel A was added after the legend was written (as the legend is shifted and missing panel C).The authors might also consider adding an additional limitation to their discussion that the lighting in the open field is different between their temperature conditions.Ideally, the lighting intensity would be the same since the conditions are being directly compared, however, as both lighting conditions are towards the high end, and the condition with lower lighting has animals exhibiting higher levels of avoidance, it is likely not a significant factor.I still have reservations about the lack of appropriate vehicle controls.The authors have added a statement in the discussion stating that DMSO is used at a dose that is welltolerated, but I feel that they should state the limitation that the effects of DMSO are not controlled for in these experiments.Pending these revisions, the report will be satisfactory.

Is the work clearly and accurately presented and does it cite the current literature?
The authors have examined Open Field behavior in different temperatures to better understand the effects of temperature fluctuations on behavior.This is an important topic that should be better explored.Here, the experiments, as described, do not appear rigorous enough to support the conclusions.Moreover, the discussion does not situate the manuscript within the wider context of the literature, nor discuss the limitations or alternative interpretations of these experiments.Main concerns are detailed below: The authors show temperature variations that are within 2-4C between seasons/rooms/buildings.However, experiments were conducted at vastly different temperatures (4C and 23C) and the conclusion drawn that "it is important to consider temperature fluctuations".The authors do not show such wild temperature fluctuations in Figure 1, so what is the rationale for examining behavior at 4C -a temperature that nearly every laboratory building will not be near (except cold rooms)?A more logical experiment (particularly in light of the authors' temperature-controlled environment) would be to measure behavior at the differing temperatures that they observe across seasons or buildings.This would better reflect the variation in the literature between universities and provide insight into conflicting data.If there was no effect of those temperatures on behavior, then it could be concluded that fluctuations within 2-4C do not affect this particular behavior.
Can the authors please discuss the rationale for their drug doses.The diazepam dose in particular has been found to be anxiogenic and potentially sedative in mice 1 .
Peripheral DMSO injections can be toxic to mice above 10%.The methods suggest that drugs were dissolved in pure DMSO and then injected i.p.Can the authors please verify that the DMSO levels were not at toxic or noxious levels?Peripheral DMSO has been shown to be painful and if drugs are administered in pure DMSO, then behavioral interpretations may be limited.
Open Field behavior is highly influenced by lighting conditions.The authors should indicate the lighting conditions of the test in lux.
In Figure 3, the authors compare the effects of temperature on open field, but present the data in separate graphs.Are the same animals tested at both temperatures?If so, the data should be analyzed by repeated measures ANOVA and the temperature considered as an additional factor to genotype.This will allow the authors to make the claim of more or less effects of cold temperature on OF behavior.
In Figure 4, are these the same groups of animals as in Figure 3? The authors should specify whether each experiment used naïve animals or those from previous experiments.The authors injected a TRPM8 antagonist but do not include vehicle-injected groups.As this drug is diluted in DMSO, this group is necessary.Moreover, the authors are making comparisons to non-injected animals in Figure 3. Statistically and experimentally, that is inappropriate unless Figure 3 animals were injected with vehicle (in which case, this needs to be properly stated and the statistics performed on all groups).
Figure 5, saline is used as the vehicle for diazepam, but again, there is no vehicle for M8-B (which the authors state is diluted in DMSO).This is inappropriate as DMSO injections evoke different reactions than saline injections.The results state that the reduction in CBT was associated with a near-significant increase in OF behavior in WT but not mutant mice, however, there are no correlations shown between behavior and temperature (did behavior change across time as CBT decreased?).Moreover, in this case, the OF was performed 30min after Diazepam administration for 1h, per the methods.Was CBT measured during the OF then?How did the authors confirm that behavior was not disrupted by this measurement?Was CBT measured at a separate time point from behavior?In which case, were the animals naïve or repeatedly injected?Finally, diazepam appears to have no effect on behavior at 4C -the authors should verify that their dose is working well at 23C to ensure that there are no confounding sedative or anxiogenic effects.
The discussion is incredibly brief and does not do anything to discuss potential limitations, explanations, or situate the manuscript within the literature.There is mention of a single review.
There are other publications that discuss the pitfalls of temperature fluctuations that should be included here.The authors need to also consider the literature of Trpm8's function and role.

Mark Zylka
The authors have examined Open Field behavior in different temperatures to better understand the effects of temperature fluctuations on behavior.This is an important topic that should be better explored.Here, the experiments, as described, do not appear rigorous enough to support the conclusions.Moreover, the discussion does not situate the manuscript within the wider context of the literature, nor discuss the limitations or alternative interpretations of these experiments.Main concerns are detailed below: The authors show temperature variations that are within 2-4C between seasons/rooms/buildings.However, experiments were conducted at vastly different temperatures (4C and 23C) and the conclusion drawn that "it is important to consider temperature fluctuations".The authors do not show such wild temperature fluctuations in Figure 1, so what is the rationale for examining behavior at 4C -a temperature that nearly every laboratory building will not be near (except cold rooms)?A more logical experiment (particularly in light of the authors' temperature-controlled environment) would be to measure behavior at the differing temperatures that they observe across seasons or buildings.This would better reflect the variation in the literature between universities and provide insight into conflicting data.If there was no effect of those temperatures on behavior, then it could be concluded that fluctuations within 2-4C do not affect this particular behavior.
>While the testing environments used in this study (23C and 4C) represent a wider temperature range than is observed between laboratories, this proof of principle study demonstrates that temperature can have an influence on behavior.More importantly, in this study we demonstrate that the body temperature changes induced following drug administration could lead to similar physiological responses induced by normal room temperature fluctuations.We have included in the discussion this limitation in interpretation.
Can the authors please discuss the rationale for their drug doses.The diazepam dose in particular has been found to be anxiogenic and potentially sedative in mice 1 .
>The drug doses were chosen based on those commonly used in the literature to study open field behavior.While we cannot eliminate the influence the drugs have on the entire body, the end goal was to make an association between behavior and body temperature.This limitation has been included in the discussion.
Peripheral DMSO injections can be toxic to mice above 10%.The methods suggest that drugs were dissolved in pure DMSO and then injected i.p.Can the authors please verify that the DMSO levels were not at toxic or noxious levels?Peripheral DMSO has been shown to be painful and if drugs are administered in pure DMSO, then behavioral interpretations may be limited.>The lighting conditions in the room temperature and cold room studies were measured at 660 lux and 400 lux, respectively.This measurement has been added to the methods section.

>DMSO
In Figure 3, the authors compare the effects of temperature on open field, but present the data in separate graphs.Are the same animals tested at both temperatures?If so, the data should be analyzed by repeated measures ANOVA and the temperature considered as an additional factor to genotype.This will allow the authors to make the claim of more or less effects of cold temperature on OF behavior.
>Yes the same animals were tested in both temperatures.The data has been analyzed using a mixed effects analysis with multiple comparisons, Sidak.Figure 3 has been updated.
In Figure 4, are these the same groups of animals as in Figure 3? The authors should specify whether each experiment used naïve animals or those from previous experiments.The authors injected a TRPM8 antagonist but do not include vehicle-injected groups.As this drug is diluted in DMSO, this group is necessary.Moreover, the authors are making comparisons to non-injected animals in Figure 3. Statistically and experimentally, that is inappropriate unless Figure 3 animals were injected with vehicle (in which case, this needs to be properly stated and the statistics performed on all groups).
>Yes, the same animals are used throughout the study except for Figure 2. The tests were conducted with a 1 week recovery period in between.This statement is included in the methods.The vehicle saline injected group is included in figure 5 for comparison.Comparison was not made to DMSO injected mice.This limitation is included in the discussion.
Figure 5, saline is used as the vehicle for diazepam, but again, there is no vehicle for M8-B (which the authors state is diluted in DMSO).This is inappropriate as DMSO injections evoke different reactions than saline injections.The results state that the reduction in CBT was associated with a near-significant increase in OF behavior in WT but not mutant mice, however, there are no correlations shown between behavior and temperature (did behavior change across time as CBT decreased?).Moreover, in this case, the OF was performed 30min after Diazepam administration for 1h, per the methods.Was CBT measured during the OF then?How did the authors confirm that behavior was not disrupted by this measurement?Was CBT measured at a separate time point from behavior?In which case, were the animals naïve or repeatedly injected?Finally, diazepam appears to have no effect on behavior at 4C -the authors should verify that their dose is working well at 23C to ensure that there are no confounding sedative or anxiogenic effects.

Figure 1 .
Figure 1.Room temperature fluctuates in lab space throughout the year if not purposefully controlled.(A) The average temperature in a semi-temperature controlled room for each month of one year (n=11-14).Temperature during the (B) winter and (C) summer measured every 20 minutes for one week within lab space, located in two different buildings and in a room that was specifically engineered to maintain temperature with minimal fluctuations over hours, days, weeks, and years (n=3-4).Temperature monitored with a La Crosse Technologies weather station.

Figure 2 .
Figure 2. Effect of icilin on CBT and open field behavior in WT mice.(A) CBT was measured every 15 minutes following 50 mg/kg bw i.p. administration of icilin (n=14) or vehicle (n=11).(B) Distance traveled and (C) time spend in the center in the open field at 23°C was measured 10 minutes post-icilin administration for one hour.Data represent means AE SEM.CBT, core body temperature; WT, wild-type; bw, body weight; i.p., intraperitoneally.

Figure 5 .
Figure 5.Comparison of open field behavior at 4°C in WT and Trpm8 -/-mice following saline (n=6), diazepam (n=8), or M8-B (n=8) administration.(A) Distance traveled and (B) center time in WT mice.(C) Distance traveled and (D) center time in Trpm8 -/-mice.Data represent means AE SEM.n=6-8 mice.WT, wild-type; Trpm8, Transient Receptor Potential Subfamily M Member 8. Data in this figure are also shown in Figures 4B and 4C and 6B and 6C.

Competing Interests: None Version 2 Reviewer
results in conclusion.Ans: Figure 6 represents the effect of diazepam on CBT and open field behavior in WT and Trpm8 knock out mice.Panel A shows the effect of diazepam on the core body temperature.Panel B shows the effect of diazepam on the open field behavior of these two genotypes at 4C. Figure 3 represents the effect of temperature on the open field behavior of WT and Trpm8 knock out mice.Mice are not injected with drugs in this experiment.Both genotypes are assessed in the open field at room temperature and in the cold room at 4C.A new graph was included in the previous revision to better represent this experiment.Report 23 January 2024 https://doi.org/10.5256/f1000research.159192.r228500© 2024 Hollis F. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Fiona Hollis 1 Department of Pharmacology, Physiology and Neuroscience, University of South Carolina School of Medicine, Columbia, SC, USA 2 Department of Pharmacology, Physiology and Neuroscience, University of South Carolina School of Medicine, Columbia, SC, USA 3 Department of Pharmacology, Physiology and Neuroscience, University of South Carolina School of Medicine, Columbia, SC, USA

Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? No Are sufficient details of methods and analysis provided to allow replication by others? No If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests:
figure legend is mislabeled.No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.
AuthorResponse 20 Nov 2023

did not exceed 2mL/kg. Studies show that mice can tolerate 10mL/kg. This limitation has been included in the discussion
. (Gad SC, Cassidy CD, Aubert N, Spainhour B, Robbe H. Nonclinical Vehicle Use in Studies by Multiple Routes in Multiple Species.International Journal of Toxicology.2006;25(6):499-521.)Open Field behavior is highly influenced by lighting conditions.The authors should indicate the lighting conditions of the test in lux.