Induction of fatigue-like behavior by pelvic irradiation of male mice alters cognitive behaviors and BDNF expression

Brian S Wolff; Sumiyya A Raheem; Sarah A Alshawi; Jeniece M Regan; Li Rebekah Feng; Leorey N Saligan

doi:10.1371/journal.pone.0235566

. 2020 Jul 2;15(7):e0235566. doi: 10.1371/journal.pone.0235566

Induction of fatigue-like behavior by pelvic irradiation of male mice alters cognitive behaviors and BDNF expression

Brian S Wolff ¹, Sumiyya A Raheem ¹, Sarah A Alshawi ¹, Jeniece M Regan ¹, Li Rebekah Feng ¹, Leorey N Saligan ^1,^*

Editor: Michelle M Adams²

PMCID: PMC7332074 PMID: 32614931

Abstract

Fatigue and cognitive deficits are often co-occurring symptoms reported by patients after radiation therapy for prostate cancer. In this study, we induced fatigue-like behavior in mice using targeted pelvic irradiation to mimic the clinical treatment regimen and assess cognitive behavioral changes. We observed that pelvic irradiation produced a robust fatigue phenotype, a reduced rate of spontaneous alternation in a Y-maze test, and no behavioral change in an open field test. We found that reversal learning for fatigued mice was slower with respect to time, but not with respect to effort put into the test, suggesting that fatigue may impact the ability or motivation to work at a cognitive task without impairing cognitive capabilities. In addition, we found that mice undergoing pelvic irradiation show lower whole-brain levels of mature BDNF, and that whole-brain proBDNF levels also correlate with spontaneous alternation in a Y-maze test. These results suggest that changes in BDNF levels could be both a cause and an effect of fatigue-related changes in behavior.

1. Introduction

Fatigue is a common symptom associated with cancer and cancer treatments, including chemotherapy and radiation therapy, and it can dramatically impact quality of life [1]. Cancer-related fatigue (CRF) often co-occurs with other symptoms, including cognitive deficits, even when the central nervous system is not directly affected by the cancer or the treatment [2, 3]. It is therefore likely that systemic changes, for example changes in immune response [4], are causing the co-occurrence of symptoms, but the process remains poorly understood. Mechanistic studies of fatigue and its co-occurring symptoms in clinical settings can be challenging, so animal models may be critical to uncover the mechanisms behind CRF and its related symptoms.

A recent study in men receiving radiation treatment for prostate cancer found that the cognitive deficits accompanying CRF were related specifically to response times and not accuracy in a cognitive task [5]. This result resembles findings in patients with chronic fatigue syndrome [6, 7] (CFS), which suggests that this specific cognitive deficit could be a common feature of fatigue across clinical populations. To our knowledge, it is unknown whether this generalizes to animal models of CRF.

Brain-derived neurotrophic factor (BDNF) is expressed throughout the brain and involved in various aspects of brain function. Mature BDNF (mBDNF) and its precursor, proBDNF, have opposing actions [8, 9]: mBDNF interacts with tropomyosin receptor kinase B (TrkB) and promotes cell survival, neurite outgrowth, and synaptic long-term potentiation; while proBDNF binds to pan-neurotrophin receptor 75 (p75^NTR) and promotes apoptosis, growth cone retraction, and synaptic long-term depression. Patients with CRF often exercise less than healthy controls [10], and exercise has been strongly associated with elevated levels of mBDNF in both clinical [4] and animal models [11, 12]. Interestingly, some beneficial effects of exercise appear to be mediated by an upregulation of BDNF in the brain, as well as an enhancement of mBDNF and a reduction of proBDNF [13]. In addition, CRF and related symptoms in human subjects have been associated not only with lower serum/plasma levels of BDNF [14, 15], but also a single-nucleotide polymorphism of the BDNF gene [16, 17]. Based on the literature, fatigue and BDNF may form a feedback loop: fatigue and its consequential decrease in voluntary activity may alter BDNF levels, and BDNF levels may also affect fatigue symptoms. The relationship between BDNF and CRF has not been well-explored, and further study is warranted to understand the connection.

The goal of the current study was to test whether we can observe a relationship between fatigue-like behavior, cognitive behavior, and BDNF expression levels in a pelvic-irradiated mouse model. It is a common question about CRF whether symptoms are driven primarily by the cancer, the treatments, or a combination of the two. Importantly, in this study we used healthy and wild-type male mice, so the effects we describe are specifically attributable to irradiation and not to any interactions with cancer. We used fractionated irradiation targeted to a pelvic region of male mice, which induces a fatigue-like behavior that is defined as a decrease in voluntary locomotor activity.

There are two main parts of the study. First, we tested whether the fatigue-like behavior is accompanied by changes in cognitive or anxiety-like behavior, and we saw significant differences in the rates of both spontaneous alternation and reversal learning when comparing irradiated and sham-irradiated mice. Second, we tested whether whole brain levels of mBDNF and proBDNF are altered by the irradiation procedure, and we found that irradiation had an effect of mBDNF levels, but that spontaneous alternation behavior correlated only with proBDNF levels.

2. Materials and methods

2.1. Ethics

This study was approved by the National Heart Lung and Blood Institute (NHLBI) Animal Care and Use Committee of the National Institutes of Health (NIH), Bethesda, Maryland, USA. All aspects of animal testing, housing, and environmental conditions used in this study were in compliance with The Guide for the Care and Use of Laboratory Animals [18].

2.2. Animals

100 six-week old male C57BL/6NCrl mice were ordered from Charles River Laboratories (Frederick MD) and were individually housed on a 12:12 hour light-dark cycle at roughly 22.2°C. All mouse handling and experimental procedures were conducted during the light cycle, and, unless otherwise specified, mice had ad libitum access to food and water. Three mice died during the study (two failed to wake up from anesthesia, the other for unknown reasons) and their data were removed from all analysis.

2.3. Irradiation

This method is described in detail in an earlier publication [19], including the design of the shielding used to target irradiation to the pelvis. In brief, mice were assigned to irradiated (“Irrad”) or sham (“Sham”) groups so that body weights were evenly distributed between groups; grouping was otherwise random. Once per day for three days, mice were anesthetized with a mixture of 100 mg/kg ketamine (MWI Animal Health, Boise, ID, USA) and 10 mg/kg xylazine (Akorn Animal Health, Lake Forest, IL, USA) and placed inside a lead shielding device within a GammaCell 40 Exactor irradiator (Best Theratronics, Ottowa Ontario, Canada), where they then received 8 Gy irradiation targeted to a pelvic region. This dose causes no overt changes in physical appearance nor signs of tissue damage in the mice [20], though it does induce a decrease in bodyweight [21]. Mice in the Sham group underwent the same procedure as those in the Irrad group, except that they were left outside of the irradiator.

2.4. Voluntary Wheel Running Activity (VWRA)

76 mice were housed in cages with running wheels (Lafayette Neuroscience, Indiana, USA) that recorded wheel rotation in one-minute intervals. Mice were transferred into running wheel cages after a week of acclimation to the animal facility. After five days of baseline VWRA recording (days -5 through -1), mice were housed in cages without running wheels for the three days of irradiation (days 0–2). The day after finishing irradiation, animals were transferred back into clean running wheel cages and VWRA was recorded for three more days (days 3–5). VWRA was quantified as the number of minutes during which the wheel rotated. We excluded data from wheels that did not accurately count revolutions or from animals that did not run a consistent amount during the baseline period, which we defined as a 50% or greater drop in recorded VWRA from one day to the next at any time during the baseline period. Data from 9 mice were excluded for these reasons.

2.5. Spontaneous arenas

The mice housed in running-wheel cages were also tested in one of two spontaneous arena tests (no mouse was tested in both). All experiments in arenas (Open Field or Y-maze) were conducted between 1 p.m. (ZT7) and 4 p.m. (ZT10). There was only a single trial in a single arena per mouse. An overhead camera recorded all trials. Distance and location information were calculated by ANY-maze software (Stoelting Co., Wood Dale, IL) and calculation of all outcome measures was automated. Illumination during the experiment was kept as low as possible while still allowing accurate tracking. The arenas were thoroughly wiped with 70% ethanol before each trial. The order of tests was balanced so that the average time of day was approximately the same between groups. The same experimenter handled the mice on the day of the test for all tests.

2.5.1. Y-maze

The Y-maze (Med Associates, Vermont, USA) was an arena with three arms (36 cm long); one arm had an additional 14 cm-long entry compartment that was separated from the rest of the maze by a removable door. Mice were placed in the entry compartment, the door was removed, and the mouse was then allowed to explore the Y-maze for 5 minutes. Scoring of arm entries was automated from the location data calculated by the software; a mouse had to be in an arm for at least one uninterrupted second for it to be considered an entry. Each new arm entry was defined as a spontaneous alternation if the two previous entries were into the two other maze arms, and the rate of spontaneous alternation was quantified as the number of spontaneous alternations divided by the total number of arm entries after the first. The test was administered on 28 mice; one mouse was removed from analysis due to making only one arm entry.

2.5.2. Open field

A mouse was placed in the center of an opaque white open field arena (45 x 45 cm) and allowed to explore for 30 minutes. A mouse was considered in the “center” of the arena if its center point was more than 10 cm from any of the walls. The test was administered on 45 mice.

2.6. Reversal learning

Mice were housed in Phenotyper cages (Noldus, Wageningen, The Netherlands), where they were recorded 24 hours per day by a camera mounted on the top of each cage. A food pellet dispenser protruded from one corner of the cage and dispensed 20 mg grain-based dustless precision pellets (Bio-serv, Flemington, NJ). Noldus Ethovision software identified nose pokes from the video, controlled the pellet dispensers, and calculated total distances travelled by the mice. The cages had a small, rectangular shelter in the corner of the cage opposite the food dispenser where the mice typically slept.

The procedure is modified from a study published by Remmelink et al [22]. During “acclimation” (days -5 through -3) and during the irradiation procedure (days 0–2), the Phenotyper cages had a food tray and no cognition wall. For “training” (days -2 and -1; also 3 and 4) the food tray was replaced by a three-holed cognition wall, situated so that mice would need to poke their nose through a hole to access food pellets. Pellets were dispensed on a fixed-ratio 5 schedule, where a pellet was dispensed after every fifth nose poke into the “correct” (left) hole. The pokes did not need to be consecutive. Reversal learning, where the “correct” hole was changed to the hole on the right, began on day 5 and continued through day 8.

The pellet dispensers were tested once per day to ensure proper function, which provided the mice one free food pellet each day. If at any point a mouse lost 10% or more of its body weight and consumed fewer than 70 food pellets, it was given enough free food pellets to bring its total up to 70 food pellets. The study started with 24 mice, but three were excluded: one did not eat, the second was due to a software error, and the third because it slept in a nose-poke position, causing an accumulation of uneaten pellets.

2.7. BDNF Western blot

Mice were anesthetized with a mixture of ketamine (120 mg/kg) and xylazine (20 mg/kg), euthanized by exsanguination, and decapitated. Whole extracted brains were flash-frozen in liquid nitrogen. Lysates were made in the same manner as previously reported [23]. The samples were denatured at 100°C for 5 minutes and loaded onto 4–20% Mini-PROTEAN^® TGX™ Precast Gels (Bio-Rad, Hercules, CA, USA). The gels were run at 100 volts and transferred to polyvinylidene difluoride membranes with Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA). Membranes were hydrated with a methanol rinse followed by a 1-hour blocking in 5% Non‑Fat Dry Milk Omniblock (AB10109-00100; AmericanBio, Inc., Canton, MA, USA) blocking buffer solution in phosphate-buffered saline with 0.1% Tween (PBST). Membranes were probed with primary antibody (anti-BDNF, 1:2,000 in blocking buffer, cat. no: ab108319; Abcam, Cambridge, UK) overnight at 4°C. After washing with PBST, the membranes were incubated with the secondary antibody (anti-rabbit IgG HRP, 1:5,000 in blocking buffer, cat. no: NA934, GE Healthcare, Chicago, IL, USA) for 1 hour at room temperature. After imaging, membranes were re-probed with a primary antibody against GAPDH (anti-rabbit, 1:1,000, cat. no: ab9485; Abcam, Cambridge, UK) as a loading control. Immunoreactive complexes were visualized using Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA), imaged with the ChemiDoc MP Imaging Systems Image Lab 6.0.1 (Bio-Rad, Hercules, CA, USA), and densitometry data was quantified with ImageJ.

2.8. Statistical analysis

Distance and pellet data were exported from Ethovision for analysis. Custom python code was used to format and plot the data and conduct statistical tests. Data were assessed for normality using a Shapiro-Wilks normality test rejecting normality at p < 0.05. A Mann-Whitney rank test was used when normality was rejected, otherwise independent two-tailed t-tests were used with an alpha level of 0.05. Effect sizes are reported as Cohen’s d. Correlations are reported as Pearson’s r. Line plots show the mean values with error bars or shaded areas showing the standard error of the mean. Box plots use matplotlib defaults, displaying the medians (center line), interquartile range (box), and the most extreme values within 1.5 times the interquartile range (whiskers). In all figures, red lines/dots/shading represent the Irrad group, and blue lines/dots/shading represent the Sham group.

3. Results

3.1. Arena tests

The timeline for running wheel and arena tests (Y-maze or open field) are shown in Fig 1A. Mice were randomized into two groups: Irrad, which underwent either three days of a targeted pelvic irradiation procedure, or Sham, which underwent three days of an identical procedure except the mice were left outside of the irradiator. On the third day after completing the irradiation procedure, each of these mice were also tested in one of two spontaneous arena tests: either a 5-minute Y-maze test or a 30-minute open field test.

Similar to previously published work [21], we found a large decrease in voluntary wheel-running activity (VWRA) in Irrad mice compared to Sham (Fig 2A). The decrease was large and statistically significant (Fig 2B, d = 1.78, t = 15.64, p < 10⁻²², n = 67). Total distance in the Y-maze was lower in Irrad animals, though the difference was not statistically significant (S2A Fig, d = 0.72, t = 1.92, p = 0.0663, n = 27). Spontaneous alternation rates were significantly lower in the Irrad animals (Fig 2C, d = 0.85, t = 2.34, p = 0.0279, n = 27), suggesting cognitive impacts of irradiation that may be related to spatial memory. Total distance in the open field test, similar to the Y-maze, was slightly but not significantly lower in Irrad animals (S2C Fig, d = 0.16, t = 0.54, p = 0.5935, n = 45). There was no significant difference in center time, a common measure of anxiety, between Irrad and Sham animals (S2D Fig, d = 0.35, t = 1.17, p = 0.2472, n = 45).

Distances in either of the two arenas showed little if any correlation with total VWRA over the three post-irradiation days (S2E & S2F Fig), suggesting that 5–30 minute arena tests are not an effective way to measure the fatigue induced by irradiation. VWRA showed a significant correlation with spontaneous alternation in the Y-maze (Fig 2D, r = 0.49, p = 0.017, n = 23), suggesting that the fatigue-like and cognitive behavioral changes may share common mechanisms. Unsurprisingly, there was no correlation between VWRA and center time in the open field test (S2G Fig).

3.2. Reversal learning

It has been previously demonstrated that in a prostate cancer population, patients experiencing fatigue after radiation therapy take longer to perform a cognitive task but do not show lower levels of performance accuracy [5]. To test whether a similar phenomenon existed in mice, we sought a cognitive test that was not time-restricted in the way that the arena tests were. We chose to test reversal learning in an automated 24-hour video recording setup. Mice were first trained to nose-poke into the left hole of a three-holed wall to receive food; the third day after completing the irradiation procedure, the rewarded hole was changed to the one on the right. Our primary outcome measure was the percentage of nose-pokes into the correct hole after it was switched for reversal learning. The mice could participate in the task as little or as much as they wanted and receive food rewards as quickly or as slowly as they wanted. The timeline of the experiment is shown in Fig 1B.

Video monitoring allowed us to record total distance travelled in the home cage, and again similar to published work on cancer-related fatigue, we found that locomotor activity was much lower the Irrad group than Sham (Fig 3A). The effect of irradiation on the total distance travelled was statistically significant (Fig 3B, d = 1.62, t = 6.17, p < 10⁻⁵, n = 21), which shows that we can still measure fatigue-like behavior while the mouse is feeding via the cognition wall. There was also a significant effect of irradiation on total food consumed (Fig 3C, d = 1.46, t = 4.71, p < 10⁻³, n = 21). There was a strong and statistically significant correlation between food consumed and distance travelled in the Irrad group (Fig 3D, r = 0.76, p = 0.006) but not the Sham group (r = 0.21, p = 0.563), suggesting that fatigue and reduced feeding may be related.

Fig 3 — “Performance” is the percentage of nose-pokes into the rewarded hole of the cognition wall. Performance line plots (E, F, H, I) display the mean performance over a 30 nose-poke rolling window. **(A)** Mean daily distances traveled. Grey shaded area indicates the three days of irradiation. **(B)** Distances travelled across the 6-day post-irradiation period normalized to the total on the day before irradiation. **(C)** Food pellets consumed per day across the 6-day post-irradiation period. **(D)** Correlation between VWRA and food consumption for each group and for the combined data. **(E)** Mean performance cumulative over the first night of reversal learning. *p < 0.05, ***p < 0.0005.

For two days after irradiation, mice continued with training and showed stable performance with little difference between groups (S5B & S5E Fig). To evaluate the subsequent reversal learning, we first calculated the performance (percent of pokes into the correct hole) as a rolling average of 30 pokes. When looking at performance with respect to time, we found no difference between groups in the initial training before irradiation (S5A Fig) but a clear difference during reversal learning after irradiation (S5C Fig). We next calculated performance over the entire first night of data collection. We chose to look at the first night because most locomotor activity and food dispensing took place at night (S4 Fig) and most of the learning (performance change) took place over the first night (S5C Fig). Comparing cumulative performance over the first night of reversal learning relative to training, the difference between groups is statistically significant (Fig 3E, d = 1.02, t = 2.65, p = 0.0167, n = 21). When looking at performance with respect to the number of pokes, we find that performance during training is again the same between groups (S5D Fig), but that the changes in reversal learning disappear (S5F & S6A Fig, d = 0.11, t = 0.24, p = 0.815, n = 21). It is clear that mice in the Irrad group are taking more time to make the equivalent number of nose pokes, suggesting that mice with irradiation-induced fatigue learn slower with respect to time primarily because they engage with a task at a slower pace. However, they do not learn slower with respect to engagement with the task.

3.3. BDNF

We next hypothesized that changes in BDNF expression in the brain may be associated with fatigue-like and cognitive behaviors. We measured both proBDNF and mBDNF in whole brain lysates from the mice described in Fig 2, which were housed with running wheels and underwent one of two arena tests. We used Western blot analysis to measure levels of mBDNF and proBDNF normalized to GAPDH (representative bands were shown Fig 4A; all raw Western Blot images used in densitometric analysis are included in S1 Raw images), and we found a significant effect of irradiation on mBDNF levels (Fig 4B, d = 0.48, U = 260.00, p = 0.0334, n = 56) but not on proBDNF (S7A Fig, d = 0.03, U = 353, p = 0.5487, n = 56). Since mBDNF expression may be upregulated by exercise, and irradiated mice exercised less on their running wheels (Fig 2A), we tested whether the change in mBDNF levels correlated with VWRA. Surprisingly, we found very little correlation (S7B Fig), which suggests that other factors, such as the irradiation process itself, may be causing the change in mBDNF levels.

Fig 4 — BDNF levels were measured in mouse whole brain lysates via Western Blot analysis three days after irradiation and one day after Y-maze or open field testing. **(A)** Representative bands from a Western blot for mBDNF, proBDNF, and GAPDH (all raw Western blot images used for densitometric analysis were included in S6 Fig). **(B)** Densitometric analysis results of mBDNF normalized the loading control, GAPDH. **(C)** Correlation between measured proBDNF levels and spontaneous alternation in the Y-maze. *p < 0.05.

Even though irradiation did not change proBDNF levels, we did find a significant negative correlation between proBDNF and spontaneous alternation performance in the Y-maze (Fig 4C, r = -0.50, p = 0.008, n = 35). This was not the case for mBDNF (S7D Fig), which suggests increased levels of proBDNF may negatively affect cognition in a way that is not related to the irradiation. This interpretation is supported by a significant correlation only in the Sham group (r = -0.86, p = 0.007, n = 14) but not the irradiated group (r = -0.49, p = 0.093, n = 19). Further, the overall correlation may be highly driven by an irradiated mouse with an unusually high level of proBDNF; when calculating the Spearman correlation, which can be more robust to outliers, correlation within the Sham group is still statistically significant (r = -0.60, p = 0.024) but the overall correlation is no longer significant (r = -0.29, p = 0.137). Open time in the open field did not significantly correlate with levels of mBDNF or proBDNF (S7E & S7F Fig).

4. Discussion

In previous studies [21], we have used a mouse model of radiation therapy for treatment of prostate cancer to show that pelvic irradiation alone is sufficient to induce a profound fatigue-like behavior that can be measured as a decline in VWRA or home cage locomotor activity. In this study, we found that this pelvic irradiation also induces a change in cognitive behavior, most notably as a decrease in spontaneous alternation in a Y-maze. We also found that the behavioral changes after irradiation are accompanied by lower levels of mBDNF in the brain.

The mechanism by which irradiation constrained to a pelvic region affects both cognitive and fatigue behaviors is unknown. In a previous study we established that weight loss is also a result of this irradiation procedure [21], so a possibility that immediately occurred to us is that reduced food consumption, as shown in Fig 3C, induces these changes. However, this may be unlikely because food restriction in rodents typically results in an increase in VWRA [24]. We would also anticipate food restriction to, if anything, show an increase in spontaneous alternation behavior, as food restriction has been shown to benefit spatial learning and memory in rodents [25] and increase BDNF and plasticity in the hippocampus [12], both of which are thought to be important for spontaneous alternation behavior [26]. A more plausible cause of behavioral changes is a systemic inflammatory response, as radiation can elevate circulating levels of pro-inflammatory cytokines [27] that may in turn alter behavior. This may resemble sickness behavior, which can include both fatigue-like behavior and cognitive deficits [28].

Exercise has been linked with spatial learning and memory as well as hippocampal neurogenesis [29], and also with an increase in hippocampal mBDNF expression [11, 30]. We initially suspected that lower levels of BDNF in the Irrad group are caused by the decrease in voluntary exercise on the running wheels. However, if this were the case, we would expect to see a correlation between VWRA and mBDNF levels, which we surprisingly did not see (S7B Fig). A future study could help clarify this relationship by using control groups of mice that are not able to exercise on running wheels. Levels of mBDNF also did not significantly correlate with spontaneous alternation rates, which is again somewhat surprising, since there is evidence that mBDNF in the hippocampus [31] and prefrontal cortex [32] is associated with improvements in spatial memory tests. However, spontaneous alternation did negatively correlate with proBDNF levels, and it has been shown previously that proBDNF in the hippocampus was associated with cognitive impairment [8]. In our study it is possible that irradiation induces signals to the CNS that shift the balance between the pro-survival and pro-apoptotic properties of mBDNF and proBDNF, respectively, which can impair cognitive abilities.

We found a negative correlation between proBDNF levels and spontaneous alternation (Fig 4C), but this result may be difficult to reconcile with other results. First, spontaneous alternation also correlated with VWRA (Fig 2D), but we did not find any correlation between proBDNF and VWRA (S7C Fig). Additionally, irradiation affected VWRA and spontaneous alternation, but did not affect proBDNF levels. It may be explained by proBDNF having an effect on spontaneous alternation that is present in the Sham group and masked by the irradiation procedure, which our results would suggest. Thus, the correlation between VWRA and spontaneous alternation is primarily driven by group differences that are not related to proBDNF. Future experiments that focus on the relationship between proBDNF and spontaneous alternation behavior may help clear this up.

Another future experiment that may be critical to elucidating the role of BDNF in radiation-induced changes in behavior would be to measure BDNF expression in different brain regions. There are reports of opposite effects of BDNF in depression-like behaviors depending on brain region, where increased BDNF in the hippocampus and frontal cortex can have antidepressant-like effects and BDNF in the ventral striatum can produce depression-like behaviors [33]. In contrast, there is evidence that BDNF in the ventral striatum can produce cognitive improvements [34], though there are not many reports of this in the literature. Since fatigue behaviors can be closely related to problems with cognition and depression, these symptoms may share distinct mechanisms related to BDNF expression.

Another important result of this study is that post-irradiation performance in the reversal learning task is impaired when evaluated with respect to time but not with respect to engagement in the task. This resembles some results seen in human subjects; for example, deficits in speed but not accuracy at cognitive tasks have been shown in CRF [5] and CFS [7] patients. We believe the most likely explanation for our results is that the willingness to engage in the task is compromised by irradiation, but the reversal learning itself is not. This would suggest that the circuitry underlying reversal learning, such as the orbitofrontal cortex and striatum [35], may be less affected by the pelvic irradiation than the circuitry associated with spontaneous alternation, for example connections between the hippocampus and temporal cortex [26].

Our understanding of the reversal learning results would benefit from future experiments that could expand on these results to distinguish the learning from related behaviors, such as attention and response times using a serial reaction time test, or motivation using a progressive ratio test. Both of those examples could be used with a similar setup to test rates versus accuracy of goal-directed behaviors. Combining these with brain-circuit-specific measures could help understand how behaviors or symptoms related to fatigue may be dissociable or overlapping.

Additionally, our reversal learning results are an important demonstration that a study where task participation is constrained by time could erroneously show cognitive deficits in fatigued participants, whether mice or human. Had our experiment design been to measure performance over one night of reversal learning, we would have reached the opposite conclusion, that there was an effect of pelvic irradiation on reversal learning. Therefore, when designing studies in which fatigue may be a factor, measurements of learning performance or accuracy should be completely independent from speed.

In conclusion, we found that in addition to inducing fatigue-like behavior, pelvic irradiation can induce impairments in cognitive behaviors, which may resemble a symptom cluster observed in humans. We also found in a reversal learning task that it takes more time for the fatigued mice to learn, but that there is no decrease in overall task performance. Finally, we find that the brains of fatigued mice show lower levels of BDNF after irradiation, and that elevation of brain proBDNF correlates with decreased cognitive performance, which suggests that changes in BDNF levels may be affecting behaviors following irradiation.

Supporting information

S1 Fig. Circadian VWRA.

Mean VWRA time active for each minute of recording, with darker colors representing more activity, light colors representing lower levels of activity, and white representing no data. Irradiation took place on days 0, 1, and 2. Zeitgeber time is the number of hours after lights are turned on at 6 a.m.

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S2 Fig. Spontaneous arena behaviors.

(A) Distance travelled in the 5-minute Y-maze test. (B) The total number of arm entries during the Y-maze were not significantly affected by irradiation (d = 0.60, t = 1.56, p = 0.132, n = 27). (C) Distance travelled in the 30-minute open field test. (D) Center time in the open field test. (E) There were no significant correlations between VWRA and distances travelled in the Y-maze. (F–G) There were no significant correlations between VWRA and distances travelled (F) or open time (G) in the open field.

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S3 Fig. Arena performance over time.

Since irradiation showed different effects on reversal learning over time vs. over participation with the task, we did a similar analysis on the arena behaviors. (A) Plotting spontaneous alternation behavior over time in the Y-maze, distance travelled, or total arm entries had little effect on the appearance of the plots. (B) Plotting center time in the open field over time in the arena or over distance traveled had little effect on the appearance of the plots. The effect size is plotted with a green line without shading and uses the right-hand axis labels.

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S4 Fig. Circadian activity during reversal learning.

(A–B) Mean locomotor distance totals for each minute of recording, with darker colors representing greater distances, light colors representing lower lesser distances, and white representing no data. (C–D) Mean number of food pellets dispensed during each minute of recording, with darker colors representing more pellets, light colors representing fewer pellets, and white representing no data. Irradiation took place on days 0, 1, and 2, and no food pellets were dispensed during this time (mice had ad libitum access to chow). Zeitgeber time is the number of hours after lights are turned on at 6 a.m.

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S5 Fig. Reversal learning behavior.

(A–C) Performance over time during training (A), retraining (B), and reversal learning (C). Dark shading represents the dark cycle (night). (D-F) Performance over the total number of pokes during training (D), retraining (E), and reversal learning (F).

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S6 Fig. Reversal learning statistics.

(A) Mean performance cumulative over the first 1536 pokes of reversal learning, which is the mean number of nose-pokes over the first night across all mice in the Sham group. (B) There were no significant correlations between distance traveled and performance during the first night of reversal learning. (C) There were large significant correlations between food pellets dispensed and performance during the first night of reversal learning, particularly for the Irrad group. This is not surprising, as performance at the task causes food pellets to dispense.

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S7 Fig. BDNF levels and behavior.

(A) Densitometric analysis results of proBDNF normalized to GAPDH. (B–C) There were no significant correlations between mBDNF or proBDNF and VWRA. (D) There were no significant correlations between mBDNF and spontaneous alternation in the Y-maze. (E–F) There were no significant correlations between mBDNF or proBDNF and open time in the open field test.

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S1 Raw images. BDNF Western blot images.

Unedited raw images from each western blot used in densitometric analyses shown in Figs 4 and S7.

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Acknowledgments

We would like to thank the NHLBI Murine Phenotyping Core for their invaluable help with experiments, including Dr. Danielle Springer, Michele Allen, Audrey Noguchi, Heather Potts, and Morteza Pieravi. We would also like to thank the NHLBI Animal Surgery and Resources Core for help with taking blood and tissue samples.

Abbreviations

CRF: cancer related fatigue
CFS: chronic fatigue syndrome
VWRA: voluntary wheel running activity
mBDNF: mature BDNF

Data Availability

All data and code are available in an OSF repository: https://osf.io/9237v/ DOI 10.17605/OSF.IO/9237V.

Funding Statement

This study was supported by the Divisions of Intramural Research of the National Institute of Nursing Research and the National Institute of Mental Health of the NIH, Bethesda, Maryland.

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PLoS One. doi: 10.1371/journal.pone.0235566.r001

Decision Letter 0

Michelle M Adams

30 Apr 2020

PONE-D-20-08431

Induction of fatigue-like behavior by pelvic irradiation of male mice alters cognitive behaviors and BDNF expression.

PLOS ONE

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Reviewer #1: The aim of this study is to clarify whether there is connection between fatique-like behavior and cognitive behavioral changes in mice and compare BDNF levels in brain.

There are several major issues with this current paper, regarding organization, data interpretation and visualization.

It is not clear why researchers used normal mice and targeted them with pelvic irradiation instead of using prostate cancer mouse model which is commercially available. If prostate cancer patients receiving radiation treatment have specific cognitive deficits with cancer-related fatigue and this needs to be investigated, the reason to use normal mice to show relationship between BDNF and CRF should be explained. Prostate cancer mouse model receiving pelvic irradiation and normal mouse received irradiation probably will give different response.

Although general structure of the manuscript is just fine, some issues must be revisited

Page2, Line 51-57, entire paragraf should be rewritten and organized.

In the entire manuscript there is no definition of BDNF. Appreviation is not described.

There is no data about weight measurement in the manuscript. During the experimention, weight loss may contribute to the behavioral assesments.

Page 3, Line 89: Is there any data for elevated 0-maze as mentioned?

Page 4, Line 132 and page 7, Line 254: There is no data for WB provided in my copy.

Page7, Line 261: It is not clear whether previous study is published or not , no reference was given. May be those data should be included in this manuscript.

Without Wb data, it is difficult to conclude the relationship BDNF level, irradiation and cognitive performance.

Reviewer #2: This is an interesting and timely study. The following issues should be addressed:

1. Please describe how shielding was performed for the localized radiation exposure. Please also indicate the details with regard to the irradiator used.

2. “Data from 8 mice were excluded from VWRA analysis due a 50% or greater drop in recorded VWRA totals during the baseline recording period.” It is not clear what this means; please explain.

3. Please refer to the time line to indicate the sequence of the behavioral tests and the interval between them in the methods section.

4. Please do not use the word “sacrificed” as standard for most journals now.

5. There are limitations to analyze whole brain levels of BDNF as more regional changes would be anticipated to play a role in wheel running and performance in the behavioral tests used in this study.

6. The reduced wheel running activity could affect BDNF levels in these animals; so what is a consequence of radiation and what is a consequence of reduced running seems hard to distinguish without a locked wheel control. This should be acknowledged and discussed.

7. It would be good to analyze whether wheel running in the light and/or dark periods was affected and whether there were circadian changes.

8. There seems no need to show the data as a percent of baseline with these striking group differences; please show actual data that is not normalized as well.

9. It seems better to only show the significant correlations and include the other data as supplementary table or figure panels.

10. The correlation shown in Figure 4F does not seem very convincing and might be driven by some individual data points.

Reviewer #3: The authors describe the effect of pelvic irradiation of male mice on cognitive behaviors and whole brain BDNF contents.

The paper showed that pelvic irradiation produced a reduced rate of spontaneous alteration in Y maze test and expression changes in mature/pro BDNF levels in whole brain. Although this study contains interesting information, mechanistic information may be less focused.

1)Why did authors examine protein BDNF levels in whole brain?

It has been reported that central BDNF exhibits opposite actions depending on the site of actions. For example, in preclinical studies, inflammation promotes reduced BDNF in the PFC and hippocampus, as well as increased BDNF in the NAc of the brain, resulting in a

depression-like phenotype in rodents (Zhang et al. Curr Neuropharmacol. 2016;14(7):721-31.)

If they try to suggest the mechanistic correlation between cognitive/emotional function and BDNF expression, it is strongly recommended to examine the BDNF levels in at least either hippocampus, PFC, or NAc in the pelvic irradiation model. Or they could additionally examine inflammatory status (ex. cytokines, microglia activation) in whole brain levels as they discussed in the Discussion (Page 7, L274: A more plausible cause of behavioral changes is a systemic inflammatory response…).

2)The Fig. 4D, 4F, 4H seems to show pro BDNF in irradiation animals. Why are the expression plots of proBDNF in each Figure so different? (The animals number could be 4D = 4F + 4H)

-It is recommended to include both irradiation (closed circle) and sham (open circle) animals in correlation plots.

-Please show the number of animals in each Figure.

3) In Fig 2I, VWRA (active time) correlates with Y maze's spotaneous alterlation. In Fig.4F, proBDNF is inversely correlated with Y maze's spotaneous alterlation. So why does VWRA not correlate with proBDNF in Fig4D? Please discuss this point.

Reviewer #4: This manuscript describes the assessment of fatigue and cognitive functions in a mouse model of pelvic irradiation. The premise of the study stems from findings in cancer patients, especially from prostate cancer patients, that fatigue is a common finding following radiation therapy and that there may or may not be an associated cognitive decline. Voluntary running wheel exercise was used before and after irradiation to assess the extent of fatigue resulting from irradiation. The mice were also assessed for working spatial memory, anxiety, reversal learning, and tissue levels of proBDNF and mBDNF in the brain. The authors observed a significant level of fatigue in the irradiated cohort which was supported by a significant reduction in voluntary wheel running activities following irradiation. Associated with the reduced voluntary activity was an impaired performance in the spontaneous alteration behavior in the Y maze exploration. Most interesting was the outcome in the reversal learning test. The authors showed that, without constrained by time, the irradiated mice were able to learn and perform to the same level of accuracy as sham controls in obtaining food pellets in the reversal phase of the nose pole/food reward learning paradigm. Consistent with the voluntary wheel running outcome, irradiated mice showed a significant reduction in ambulatory activities in the reversal learning arena, and the reduced activity correlated with reduced food intake. The authors also observed a significant reduction of mBDNF in the brains of irradiated mice, which may be a result of reduced overall physical activity. The studies are carefully designed and the results are relevant to a significant problem in cancer patients. The study results should be of interest to clinician and investigators in the radiation and cancer therapy field in general. Several issues warrant further clarification from the authors.

1. Given that importance of the results from reversal learning, it would have been nice to have a separate test to confirm the results. In the absence of such confirmation, authors should discuss what future studies may be employed to strengthen the conclusion in the Discussion.

2. It was not clear initially that two separate sets of mice were used for the voluntary wheel running/OF/Y maze and the reversal learning and that mice in the voluntary wheel running group only went through OF or Y maze, but not both arena tests. Providing these information earlier in the Methods section would have been helpful.

3. Elevated zero maze was not performed (or at least the data were not provided), but was mentioned in line 89.

4. There was a mention OF and Y maze tests were performed between ZT7 and ZT10 (line 89-90). Please clarify what is ZT7 and ZT10.

5. The reversal learning started on Day 5 (or 3 days after irradiation), but it was not clear how many days were devoted to the reversal phase. Was there a proficiency criterion to reach before the test was terminated?

6. As a related question – there were two more days of training after irradiation. Were the performance from the irradiated mice comparable to the sham controls, and how was the performance compared to the pre-irradiation phase?

7. To measure performance without time constraint, the authored chose to analyze performance during the first 1600 nose poke in the reversal phase. What was the rationale for selecting 1600 nose pokes as the cutoff?

8. It will be helpful if the authors can indicate on the timescale (Figures E and F) which segments are the dark phase and which are the light phase. Likewise, because the time required to complete 1600 nose pokes in the reversal phase is different between sham and irradiated animals, it will be helpful for the authors to provide that information.

9. Figures 3G and 3J are a percentage over a percentage (performance during the reversal phase over performance during the first night of training). It is also not clear if 3J uses the average performance over the entire reversal phase, or just during the first 1600 nose pokes, and normalizes it to the first 1371 nose pokes. It may be helpful in visualizing the data if the authors can plot out the performance during the first night or the first 1600 nose pokes of the reversal learning and performance of the first night or the first 1371 nose pokes, respectively, during the training phase for side-by-side comparisons.

10. The authors put sample sizes in 2A and 3A, but did not provide the same information for Y maze, OF, and BDNF plots. For consistency, these information should be provided.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: Yes: Ting Ting Huang

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PLoS One. 2020 Jul 2;15(7):e0235566. doi: 10.1371/journal.pone.0235566.r002

Author response to Decision Letter 0

29 May 2020

Reviewer #1

The aim of this study is to clarify whether there is connection between fatique-like behavior and cognitive behavioral changes in mice and compare BDNF levels in brain.

There are several major issues with this current paper, regarding organization, data interpretation and visualization.

1. It is not clear why researchers used normal mice and targeted them with pelvic irradiation instead of using prostate cancer mouse model which is commercially available. If prostate cancer patients receiving radiation treatment have specific cognitive deficits with cancer-related fatigue and this needs to be investigated, the reason to use normal mice to show relationship between BDNF and CRF should be explained. Prostate cancer mouse model receiving pelvic irradiation and normal mouse received irradiation probably will give different response.

Authors: This is an important point for us to address, and we thank you for raising it. We used a normal mouse model instead of a prostate cancer mouse model to investigate the behavioral effects specific to irradiation. It is not clear from clinical populations what fatigue-like effects are due to cancer, what effects are due to treatment, and what effects are caused by the interaction of the two. We have added this information into the Introduction section.

2. Although general structure of the manuscript is just fine, some issues must be revisited

Page2, Line 51-57, entire paragraf should be rewritten and organized.

Authors: We have reworded this paragraph to make it clearer.

3. In the entire manuscript there is no definition of BDNF. Appreviation is not described.

Authors: We have defined the abbreviation of BDNF the first time it is used in the introduction.

4. There is no data about weight measurement in the manuscript. During the experimention, weight loss may contribute to the behavioral assesments.

Authors: This is correct and an important point. We did not include data on body weight in this manuscript. Weight loss occur after this irradiation procedure, as described in previous work (Wolff et al., Scientific Reports, 2018) cited in the manuscript. This weight loss may contribute to changes in behaviors, and we discussed food consumption in the second paragraph of our original discussion section. In this revised draft, we added a note about weight loss to more thoroughly address this important point.

5. Page 3, Line 89: Is there any data for elevated 0-maze as mentioned?

Authors: We apologize, this was an editing error on our part and the mention of elevated 0-maze is removed.

6. Page 4, Line 132 and page 7, Line 254: There is no data for WB provided in my copy.

Authors: This was an unfortunate omission, thank you for pointing it out. Western blot data have been added to Figure 4 (representative bands) and supplemental Fig S6, which includes all raw, unedited Western Blot images used for densitometric analysis.

7. Page7, Line 261: It is not clear whether previous study is published or not, no reference was given. May be those data should be included in this manuscript.

Authors: These data are published, so the reference has been added.

8. Without Wb data, it is difficult to conclude the relationship BDNF level, irradiation and cognitive performance.

Authors: The Western blot data have been added to Figure 4 (representative plot) and supplemental Fig S6, which includes all raw, unedited Western Blot images used for densitometric analysis.

Reviewer #2

This is an interesting and timely study. The following issues should be addressed:

1. Please describe how shielding was performed for the localized radiation exposure. Please also indicate the details with regard to the irradiator used.

Authors: We have added the irradiator model into the methods. The shielding is described in detail in Wolff et al., JoVE 2017. Rather than duplicate the published description, we have clarified that shielding details are contained in the cited publication.

2. “Data from 8 mice were excluded from VWRA analysis due a 50% or greater drop in recorded VWRA totals during the baseline recording period.” It is not clear what this means; please explain.

Authors: This has been clarified in the text, and we also fixed an incorrect count (there were actually 9 excluded animals.

3. Please refer to the time line to indicate the sequence of the behavioral tests and the interval between them in the methods section.

Authors: We regret the confusion on this point. Since multiple reviewers mentioned this, we clarified that mice underwent only one of the two behavioral tests in the methods section, results section, and in the legend for Figure 1.

4. Please do not use the word “sacrificed” as standard for most journals now.

Authors: We have changed this to “euthanized”.

Authors: We agree with this and have added a paragraph to the discussion to discuss regional changes.

Authors: This is true, and a very important point that the exercise may be a primary cause of different BDNF levels. We did not see a correlation between wheel running and BDNF levels, but using the control groups you mentioned would be a more convincing experiment. We have edited our discussion of this topic in the third paragraph of the Discussion section to make this point clear.

7. It would be good to analyze whether wheel running in the light and/or dark periods was affected and whether there were circadian changes.

Authors: We did an analysis of circadian changes in VWRA in a previous publication (Wolff et al., Scientific Reports, 2018). We have added a figure (Figure S1) to the supplemental material to show that this study is consistent with earlier results.

8. There seems no need to show the data as a percent of baseline with these striking group differences; please show actual data that is not normalized as well.

Authors: We have changed most of the figures to show non-normalized data. We would agree with the reviewer our absolute measures (e.g. spontaneous alternation or BDNF levels) should be analyzed alongside an absolute measure of physical activity.

Figures 2B and 3B still show normalized data, as we believe this is the most important measure of “fatigue”. Locomotor activity levels at baseline shows a lot of variability from animal to animal, presumably because some animals prefer to move around more than others. The sources of this variability will likely be present after irradiation as well, meaning we expect the animals that move more before irradiation to also move more after irradiation. We are not interested in this baseline tendency for locomotor activity; what we are interested in is how much this baseline tendency changes. This change in activity (normalized data), rather than the absolute level of activity, is what we consider to be the direct outcome of irradiation. However, figures 2A and 3A display the non-normalized data in order to show that the groups have similar values at baseline.

9. It seems better to only show the significant correlations and include the other data as supplementary table or figure panels.

Authors: Non-significant correlation data have been moved to the supplementary materials.

10. The correlation shown in Figure 4F does not seem very convincing and might be driven by some individual data points.

Authors: It does look like the data point furthest to the right is driving a lot of the correlation. We have noted this in the results section, and we added analysis of the correlation within individual groups. Doing so shows that the correlation only looks robust within the Sham group.

Reviewer #3

The authors describe the effect of pelvic irradiation of male mice on cognitive behaviors and whole brain BDNF contents.

1. Why did authors examine protein BDNF levels in whole brain?

depression-like phenotype in rodents (Zhang et al. Curr Neuropharmacol. 2016;14(7):721-31.)

Authors: Thank you for raising this point. These are very good ideas, and we have added a paragraph to the discussion about this.

2. The Fig. 4D, 4F, 4H seems to show pro BDNF in irradiation animals. Why are the expression plots of proBDNF in each Figure so different? (The animals number could be 4D = 4F + 4H)

-It is recommended to include both irradiation (closed circle) and sham (open circle) animals in correlation plots.

-Please show the number of animals in each Figure.

Authors: We agree this was confusing, and we are now displaying the number of animals in each figure. The Irrad and Sham groups are displayed separately in red and blue, and we have added correlation analysis for both the overall data set and for the individual groups. We thank you for raising this point, as it has really improved the quality of the manuscript.

The values for pro-BDNF displayed in the figures mentioned are almost all the same, with a few differences due to missing data. Most obviously, there is a pro-BDNF value of about 7 that appears in spontaneous alternation correlation plot (now Fig 4D), but not in the running wheel correlation plot (now Fig S5B). The running wheel data for this mouse were excluded due to a technical malfunction, where the running wheel was still spinning but not recording data. Because the running wheel was still spinning normally, we included this mouse’s Y-Maze and BDNF data. There were missing data points in each data set, which were described in the methods section, and therefore the sample size numbers don’t always match when comparing one data set to another.

Please note that at another reviewer’s request we have moved non-significant correlation plots to the supplementary data, so the Figure labels (e.g. 4A, 4B, etc.) have changed from the previous version.

3. In Fig 2I, VWRA (active time) correlates with Y maze's spotaneous alterlation. In Fig.4F, proBDNF is inversely correlated with Y maze's spotaneous alterlation. So why does VWRA not correlate with proBDNF in Fig4D? Please discuss this point.

Authors: You are correct, and we agree that this is worth discussing. We have added a paragraph to the Discussion addressing this point.

Reviewer #4

This manuscript describes the assessment of fatigue and cognitive functions in a mouse model of pelvic irradiation. The premise of the study stems from findings in cancer patients, especially from prostate cancer patients, that fatigue is a common finding following radiation therapy and that there may or may not be an associated cognitive decline. Voluntary running wheel exercise was used before and after irradiation to assess the extent of fatigue resulting from irradiation. The mice were also assessed for working spatial memory, anxiety, reversal learning, and tissue levels of proBDNF and mBDNF in the brain. The authors observed a significant level of fatigue in the irradiated cohort which was supported by a significant reduction in voluntary wheel running activities following irradiation. Associated with the reduced voluntary activity was an impaired performance in the spontaneous alteration behavior in the Y maze exploration. Most interesting was the outcome in the reversal learning test. The authors showed that, without constrained by time, the irradiated mice were able to learn and perform to the same level of accuracy as sham controls in obtaining food pellets in the reversal phase of the nose pole/food reward learning paradigm. Consistent with the voluntary wheel running outcome, irradiated mice showed a significant reduction in ambulatory activities in the reversal learning arena, and the reduced activity correlated with reduced food intake. The authors also observed a significant reduction of mBDNF in the brains of irradiated mice, which may be a result of reduced overall physical activity. The studies are carefully designed and the results are relevant to a significant problem in cancer patients. The study results should be of interest to clinician and investigators in the radiation and cancer therapy field in general. Several issues warrant further clarification from the authors.

Authors: We agree, and we have added a short paragraph to the Discussion about experiments to follow up on these results.

Authors: We regret the confusion on this point. Since multiple reviewers mentioned this point, we clarified in the methods section, results section, and in the legend for Figure 1 that mice underwent only one of the two behavioral tests.

3. Elevated zero maze was not performed (or at least the data were not provided), but was mentioned in line 89.

Authors: We apologize, this was an editing error on our part and the mention of elevated 0-maze is removed.

4. There was a mention OF and Y maze tests were performed between ZT7 and ZT10 (line 89-90). Please clarify what is ZT7 and ZT10.

Authors: We have clarified this in the methods and to the legends for Figs S1 and S3.

Authors: We regret leaving this out of the methods section, so we have added that reversal learning terminated after day 8. We did not use a proficiency criterion (we generally wish to avoid arbitrary thresholds) so we recorded for 4 days, which based on published data we expected to be more than enough time to completely capture reversal learning.

Authors: We have added supplementary figures (Figs S4A and B) to display this data. We also added a note in the results section that the performance appears similar between groups and also similar to performance at the end of the pre-irradiation phase.

Authors: We regret the lack of clarity here in the original manuscript. For Figs 3H and 3I, we chose 1600 pokes as an arbitrary axis limit for displaying the data, but as it may be confusing, we are now displaying the entire data set on these line plots. Figure 3G shows performance over the first night. For Figure 3J, we analyze the first 1536 pokes because this was the average (mean) number of pokes across all Sham animals during the first night.

Authors: The irradiated animals take longer to make an equivalent number of pokes during reversal learning, so we have clarified this in the text of the Results section. We have also added shaded regions to Figs 3E and F to indicate the dark phase. In doing so, we should mention that in the original submission of this manuscript the data were not properly aligned to one another due to video recordings starting at slightly different times of day. We have now realigned the data appropriately, which changes the numbers slightly, but does not affect our interpretation of the data, nor does it affect the results of any significance tests.

Authors: We regret the confusion on this point. To address this, we are no longer normalizing the data; instead we are just showing the raw performance (there is no longer a percentage over a percentage). We have also done our best to clarify that Fig 3G shows the cumulative performance over the first night, and Fig 3J show the cumulative performance over the first 1536 nose-pokes (which is the average number of nose pokes over the first night). We hope this sufficiently clarifies the results.

10. The authors put sample sizes in 2A and 3A, but did not provide the same information for Y maze, OF, and BDNF plots. For consistency, these information should be provided.

Authors: We have added sample sizes to all figures.

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PLoS One. doi: 10.1371/journal.pone.0235566.r003

Decision Letter 1

Michelle M Adams

15 Jun 2020

PONE-D-20-08431R1

Induction of fatigue-like behavior by pelvic irradiation of male mice alters cognitive behaviors and BDNF expression.

PLOS ONE

Dear Dr. Saligan,

As you will note Reviewer 2 has some additional minor comments that need to be addressed before the manuscript can be accepted. When the corrections are made, they will be reviewed by me and there is no need for additional review.

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We look forward to receiving your revised manuscript.

Kind regards,

Michelle M. Adams, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

I have also received Reviewer 1's comments indicating that the manuscript is now suitable for acceptance.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Yes

Reviewer #3: (No Response)

Reviewer #4: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: (No Response)

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

Reviewer #3: (No Response)

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

Reviewer #3: (No Response)

Reviewer #4: Yes

**********

6. Review Comments to the Author

Reviewer #2: While the authors have been responsive, the following changes are required to the figures:

1. Fig. 2. remove panels C, E, and F to suppl data as there is no significance; put the number of mice in the figure legend; in the G panel, show only the significant correlation and mention the other two in the text or figure legend.

2. Fig. 3. remove panels E, F, H, I, J to suppl data as there is no significance; put the number of mice in the figure legend; in the D panel, show only the two significant correlation and mention the third one in the text or figure legend.

3. Fig. 4. remove panel C to suppl data; panel D, show only the significant correlations; put the mouse numbers in the figure legend.

Reviewer #3: (No Response)

Reviewer #4: The authors have satisfactorily addressed all comments. There are two minor issues that require authors' attention:

1. Figure 3C, Y axis. This is normalized data (normalized to food pellets consumed on the day before irradiation). Consequently, the Y axis should be labeled as Food Pellets (% baseline).

2. Figure S5 D&E. Here the authors label Y axis as Open Time. Is this time in the center of the OF arena? If so, Center Time should be used to be consistent with labels used in Figures 2 and S2.

3. Figure S3. Can the authors provide a scale bar for the color scheme? The descriptive language "with darker colors representing more pellets, light colors representing fewer pellets" is not very helpful.

**********

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Reviewer #2: No

Reviewer #3: No

Reviewer #4: Yes: Ting-Ting Huang

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Jul 2;15(7):e0235566. doi: 10.1371/journal.pone.0235566.r004

Author response to Decision Letter 1

17 Jun 2020

Reviewer #2: While the authors have been responsive, the following changes are required to the figures:

3. Fig. 4. remove panel C to suppl data; panel D, show only the significant correlations; put the mouse numbers in the figure legend.

Authors: We have made the requested changes, moving non-significant results to supplementary material and removing non-significant correlation values (r and p values) from the figures (though they are still written in the main text of the Results section). We have added the number of mice to the figure legend of Fig 1, where we describe the experimental design; all other figures already show the number of mice (which was requested by one of the other reviewers in the previous round of comments). The number of mice is also written in the text of both the Methods section and in the Results section.

Reviewer #4: The authors have satisfactorily addressed all comments. There are two minor issues that require authors' attention:

1. Figure 3C, Y axis. This is normalized data (normalized to food pellets consumed on the day before irradiation). Consequently, the Y axis should be labeled as Food Pellets (% baseline).

Authors: Thank you for pointing this out. The Y-axis was correct, but the figure legend was wrong, and so we have corrected the legend. To be clear, the data are not normalized; mice often eat around 100 pellets per day.

2. Figure S5 D&E. Here the authors label Y axis as Open Time. Is this time in the center of the OF arena? If so, Center Time should be used to be consistent with labels used in Figures 2 and S2.

Authors: You are correct, and we have changed the axis label to “Center time” and expressed it properly as a percentage.

Authors: We agree, and we have added a scale bar to the all the figures that are in this format. We also reworded the figure legends to be more clear about what we measured.

Attachment

Submitted filename: reviewer comments v01.docx

Click here for additional data file.^{(19.7KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0235566.r005

Decision Letter 2

Michelle M Adams

18 Jun 2020

Induction of fatigue-like behavior by pelvic irradiation of male mice alters cognitive behaviors and BDNF expression.

PONE-D-20-08431R2

Dear Dr. Saligan,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Michelle M. Adams, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0235566.r006

Acceptance letter

Michelle M Adams

23 Jun 2020

PONE-D-20-08431R2

Induction of fatigue-like behavior by pelvic irradiation of male mice alters cognitive behaviors and BDNF expression.

Dear Dr. Saligan:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Michelle M. Adams

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Circadian VWRA.

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S2 Fig. Spontaneous arena behaviors.

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S3 Fig. Arena performance over time.

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S4 Fig. Circadian activity during reversal learning.

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S5 Fig. Reversal learning behavior.

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S6 Fig. Reversal learning statistics.

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S7 Fig. BDNF levels and behavior.

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S1 Raw images. BDNF Western blot images.

Unedited raw images from each western blot used in densitometric analyses shown in Figs 4 and S7.

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Data Availability Statement

All data and code are available in an OSF repository: https://osf.io/9237v/ DOI 10.17605/OSF.IO/9237V.

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PERMALINK

Induction of fatigue-like behavior by pelvic irradiation of male mice alters cognitive behaviors and BDNF expression

Brian S Wolff

Sumiyya A Raheem

Sarah A Alshawi

Jeniece M Regan

Li Rebekah Feng

Leorey N Saligan

Roles

Abstract

1. Introduction

2. Materials and methods

2.1. Ethics

2.2. Animals

2.3. Irradiation

2.4. Voluntary Wheel Running Activity (VWRA)

2.5. Spontaneous arenas

2.5.1. Y-maze

2.5.2. Open field

2.6. Reversal learning

2.7. BDNF Western blot

2.8. Statistical analysis

3. Results

3.1. Arena tests

Fig 1. Experiment design.

Fig 2. Arena tests.

3.2. Reversal learning

Fig 3. Reversal learning.

3.3. BDNF

Fig 4. BDNF levels.

4. Discussion

Supporting information

Acknowledgments

Abbreviations

Data Availability

Funding Statement

References

Decision Letter 0

Michelle M Adams

Roles

Author response to Decision Letter 0

Decision Letter 1

Michelle M Adams

Roles

Author response to Decision Letter 1

Decision Letter 2

Michelle M Adams

Roles

Acceptance letter

Michelle M Adams

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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