Individual Differences in Attentional Deficits and Dopaminergic Protein Levels following Exposure to Proton Radiation Author(s): Catherine M. Davis , Kathleen L. DeCicco-Skinner , Peter G. Roma and Robert D. Hienz Source: Radiation Research, 181(3):258-271. 2014. Published By: Radiation Research Society DOI: http://dx.doi.org/10.1667/RR13359.1 URL: http://www.bioone.org/doi/full/10.1667/RR13359.1

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RADIATION RESEARCH

181, 258–271 (2014)

0033-7587/14 $15.00 Ó2014 by Radiation Research Society. All rights of reproduction in any form reserved. DOI: 10.1667/RR13359.1

Individual Differences in Attentional Deficits and Dopaminergic Protein Levels following Exposure to Proton Radiation Catherine M. Davis,a,1 Kathleen L. DeCicco-Skinner,b Peter G. Romaa,c and Robert D. Hienza a

Division of Behavioral Biology, Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland; b Department of Biology, American University, Washington, DC; and c Institutes for Behavior Resources, Baltimore, Maryland

INTRODUCTION Davis, C. M., DeCicco-Skinner, K. L., Roma, P. G. and Hienz, R. D. Individual Differences in Attentional Deficits and Dopaminergic Protein Levels following Exposure to Proton Radiation. Radiat. Res. 181, 258–271 (2014).

Future long-duration space missions will involve travel outside of the protection of Earth’s magnetosphere and thereby greatly increase astronauts’ exposure to space radiation (1). In addition to the potential for changing an astronaut’s risk for cancer in later life (2), such exposure could permanently damage multiple tissues, including the central nervous system (CNS) and result in deleterious effects on the brain and behavior. Ground-based studies already indicate that exposure to space radiation in the form of protons or high-energy charged particles (HZE particles) can induce profound neurobehavioral changes in rodents. Exposure to 56Fe particles, the densest HZE ion present in cosmic rays (3), can intensify the degeneration of hippocampal-dependent memory (4), accelerate deficiencies in spatial learning and memory (5, 6), reduce hippocampal neurogenesis (7), impair spatial learning (8) and attentional set shifting (9) and produce structural alterations in brain regions important for memory consolidation such as the entorhinal cortex and thalamus (10). 56Fe particles, gamma rays and protons can also produce deficits in several neurotransmitter systems, including dopamine-mediated behaviors such as amphetamine-induced conditioned taste aversion learning (11–13), conditioned place preference (14, 15), motor performance (16, 17), sensitivity to haloperidolinduced catalepsy (18), changes in oxotremorine-enhanced K( þ)-evoked dopamine release (17, 19), alterations in glutamate transport in neurons and astrocytes (20), and preand postsynaptic changes in glutamate signaling (21). Though these findings underscore the potential dangers of radiation exposure, there still remains a minimal understanding of the likely neurobehavioral consequences to astronauts after exposure to this special form of radiation, particularly its long-term effects over extended periods of time. In an effort to determine the likely CNS effects of space radiation in astronauts during future long-duration missions, we employed behavioral procedures with rats that are, readily generalizable to humans in terms of neurobehavioral function, direct counterparts to human neurobehavioral tests, and clinically relevant for human neurobehavioral assessments. Thus, these procedures are of particular

To assess the possible neurobehavioral performance risks to astronauts from living in a space radiation environment during long-duration exploration missions, the effects of head-only proton irradiation (150 MeV/n) at low levels (25–50 cGy, approximating an astronaut’s exposure during a 2-year planetary mission) were examined in adult male Long-Evans rats performing an analog of the human psychomotor vigilance test (PVT). The rodent version of PVT or rPVT tracks performance variables analogous to the human PVT, including selective attention/ inattention, inhibitory control (‘‘impulsivity’’) and psychomotor speed. Exposure to head-only proton radiation (25, 50, 100 or 200 cGy) disrupted rPVT performance (i.e., decreased accuracy, increased premature responding, elevated lapses in attention and slowed reaction times) over the 250 day testing period. However, the performance decrements only occurred in a subgroup of animals at each exposure level, that is, the severity of the rPVT performance deficit was unrelated to proton exposure level. Analysis of brain tissue from irradiated and control rats indicated that only rats with rPVT performance deficits displayed changes in the levels of the dopamine transporter and, to a lesser extent, the D2 receptor. Additional animals trained to perform a line discrimination task measuring basic and reversal learning showed no behavioral effects over the same exposure levels, suggesting a specificity of the proton exposure effects to attentional deficits and supporting the rPVT as a sensitive neurobehavioral assay. Ó 2014 by Radiation Research Society

1 Address for correspondence: Division of Behavioral Biology, Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Bayview Campus-BBRC Suite 3000, 5510 Nathan Shock Drive, Baltimore, MD 21224; e-mail: cdavis91@ jhmi.edu.

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importance for assessing the human CNS consequences of radiation exposure. The main procedure employed was an animal counterpart to the human psychomotor vigilance test or PVT. Originally developed by Dinges et al. (22–29) as a human cognitive neurobehavioral assay for tracking temporally dynamic changes in sustained attention, the PVT has been widely validated and shown to be sensitive to sleep loss, circadian misalignment, drug use and age (24, 30). The PVT has been used to quantify risk assessment in a range of operational environments (e.g., military, aviation and railway industries, first responders) (31) and is also employed in extreme environments such as NASA’s Extreme Environment Missions Operations (NEEMO), the international MARS500 Project (32) and on the International Space Station (ISS) where it is referred to as the ‘‘Reaction Self Test’’ by astronauts and provides them with individualized performance feedback. The rodent version of the PVT (rPVT), is a simple reaction time task (SRT) in which rats are trained to respond on a nose-poke key when a light behind the key is randomly illuminated. SRT procedures have been used for decades in animal research to examine a variety of sensory and motor functions (33–44). What distinguishes the rPVT in the current study from other SRT procedures is: (1) the use of procedure parameters that are parallel to those used in human PVT research; and (2) the use of the same dependent measures as in the human PVT to track performance accuracy, attention/inattention, premature responding (‘‘impulsivity’’) and motor speed (45, 46). The validity of the rPVT to mimic human PVT performance decrements has already been shown in studies of sleep deprivation and fatigue in rats (45, 46). In the current study, adult male Long-Evans rats were trained in the rPVT and/or a visual line orientation discrimination (LD) task that required rats to learn to discriminate vertical from horizontal line stimuli and then to learn the reverse discrimination. The LD task measures the rapidity with which an animal can adjust to changes in the stimuli associated with reinforcement (47), and was used in the current study as an aid in determining the specificity of any radiation-induced deficits relative to the rPVT task. Both tasks were first trained in the laboratory until baseline performances stabilized. The rats were then transported to Brookhaven National Laboratory where they were exposed to head-only proton radiation, then returned to our laboratory for daily behavioral testing to determine the immediate and long-term effects of proton exposure on neurobehavioral performance. The current article details individual differences in rPVT performance after irradiation and includes analyses that identify and confirm the existence of a subgroup of ‘‘radiation-sensitive’’ rats based on rPVT performance measures. Given the deficits apparent in rPVT performance and the important role that dopamine (DA) plays in higher cognitive functions such as attention, working memory and inhibitory control, levels of the dopamine transporter (DAT) and D2 receptor were also

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assessed in these rats to determine whether a biological correlate of these individual differences existed. MATERIALS AND METHODS Subjects and Apparatus Subjects were 107 male Long-Evans rats (Harlan Laboratories, Indianapolis, IN) acquired at approximately 10–12 weeks of age. Rats were housed in individual plastic cages and maintained on a 12:12 h light/dark cycle (lights on at 6:00 a.m.) and at an ambient temperature of 238C for the duration of the experiment. Rats were run on their respective behavioral task during their light-on cycle at the same time each day in identically constructed operant chambers. Each rPVT chamber contained two nose-poke keys, cue lights, a house light and a food cup for delivery of food pellets. Each LD chamber was equipped with one touch screen encompassing the entire front panel and a food cup for delivery of food pellets located in the center of the opposite wall. All chambers were contained in sound-attenuating enclosures equipped with a speaker and an exhaust fan. Both the rPVT and LD sessions were 30 min in duration. The weights of the rats were permitted to increase to about 335 g 6 5 g and were then maintained within that range by feeding measured amounts of rat chow about the same time each day (30 min after the experimental session, 5 days/ week). Water was freely available in the home cage. Extra food was also provided on weekends or when no experimental sessions were scheduled. For the rPVT procedure, experimental contingencies were controlled by MedPCt behavioral control programs running on personal computers. The programs recorded all data on a trial-by-trial basis to provide for a wide range of subsequent analyses. For the LD procedure, experimental contingencies were controlled by WhiskerControlt and MonkeyCantabt behavioral control programs that ran on a Pentium III personal computer. Laboratory animal care was according to Public Health Service (PHS) Policy on the Humane Care and Use of Laboratory Animals, and the Institutional Animal Care and Use Committee of the Johns Hopkins University approved all procedures. Johns Hopkins also maintains accreditation of their program by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC). Rodent Psychomotor Vigilance Test (rPVT) A total of 41 rats (i.e., rPVT only rats, see Table 1) were first trained to respond on a nose-poke key for food pellets on a fixed-ratio (FR) 1 schedule of reinforcement. Once this behavior was acquired, training on the rPVT procedure was initiated. Sessions began with the onset of the house light. After a variable delay of 3–10 s a light behind the nose-poke key was illuminated. A correct response was defined as a response on the nose-poke key within 1.5 s after the light onset (i.e., 1.5 s limited hold, LH) and was reinforced with a pellet, while a poke prior to the light onset (premature response) or after the 1.5 s interval had elapsed (miss) was not reinforced. The delay period for the next trial began after a 3 s intertrial interval, timed either after the response or at the end of the 1.5 s LH, whichever occurred first. Data collected were the numbers of correct responses as defined above, premature responses (responses that occurred prior to the light onset), misses (number of 1.5 s light-on intervals during which no response occurred) and lapses in responding (misses plus responses greater than twice the mean response latency). Summary measures were expressed as percent as follows: accuracy ¼ correct responses/(corrects þ premature responses þ misses); premature responding ¼ number of premature responses/(corrects þ premature responses þ misses); lapse rate ¼ (misses þ lapses)/(corrects þ premature responses þ misses). In the literature on human PVT performance, lapses are considered an important indicator of inattention and/or fatigue and are typically defined as a response with a latency greater than 500 ms or roughly twice the average latency for humans performing the 10 min version

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TABLE 1 Experimental Design Radiation dose (cGy) 200 100 50 25 0 Total

Behavioral procedure and n rPVT

rPVT þ LD

LD

Total n

10 8 8 9 6 41

4 0 6 0 5 15

10 10 10 10 11 51

24 18 24 19 22 107

of the test. For rodents, average latency can vary considerably from subject to subject, and so the definition adopted here was based on each rat’s individual mean latency measure. Premature responding was broken down further by calculating a false alarm (FA) rate for those premature responses occurring within the 3–10 s delay period (i.e., FA rate ¼ premature responses within the 3–10 s delay interval)/(corrects þ premature responses within the 3–10 s delay interval þ misses). The addition of a false alarm measure allowed for the calculation of a d 0 index of signal discriminability in which percent correct (PC) scores and FA rates are converted into z scores and subtracted [d 0 ¼ z(PC) – z(FA) (48)]. Finally, response latencies to the light onset were recorded in milliseconds and summarized by calculating both the median and mean latency values. The criterion for inclusion in the current study was that rats achieve at least 75% response accuracy and less than a 25% false alarm rate for all session trials in four out of the five daily test sessions during the two weeks prior to irradiation. These criteria produced a d 0 signal detection index of 1.35, which indicates a clear discrimination above chance. Most rats achieved this criterion more than two weeks prior to irradiation, and the average pre-exposure d 0 index achieved was 2.63 (accuracy of 85.2%, false alarm rate of 5.7%). Thus, all rats included in the study acquired and maintained stable rPVT performances prior to exposure. To assign rats to radiation dose groups, a pseudorandom ranking technique was used. Specifically, a d 0 index was calculated for each rat across the five sessions completed during the week prior to irradiation. Animals were ranked from highest score to lowest score and were then assigned to one of the five dose groups (0, 25, 50, 100 or 200 cGy) such that the average d 0 index was comparable across groups.

as a missed trial. Following a correct response there was a 5 s intertrial interval, while following an incorrect choice there was a 9 s intertrial interval. Left and right positions of the S þ and S– stimuli were varied trial by trial in a pseudorandom fashion, and each session consisted of a maximum of 200 correct trials or 30 min, whichever occurred first. Rats were required to achieve 70% or greater in correct responses to the S þ during a session to complete the discrimination stage and move on to the reversal discrimination during subsequent sessions (i.e., the former S– stimulus was now the S þ and the former S þ stimulus was now the S– stimulus). In this type of procedure, random responding results in a 70% or higher accuracy rate at a probability of P ¼ 0.0001 (52). Rats remained on the reversal discrimination until the same criterion was met (i.e., 70% correct responding on the new S þ). The basic measure for assessing reversal learning was the speed of reversal learning, as indicated by the total number of trials to achieve the criterion (completed over multiple sessions, if needed). In assigning LD rats to radiation dose groups, rats were ranked on their performances during the stimulus reversal portion of the procedure by calculating the number of trials required to achieve the discrimination criterion for each rat prior to radiation. Animals were ranked from lowest to highest number of trials and were then randomly assigned to one of the five dose groups (0, 25, 50, 100 and 200 cGy) such that the average number of trials to criterion was comparable across groups. Combined rPVT and LD Tests A total of 15 rats (i.e., rPVT þ LD task rats, see Table 1) were trained to perform both the rPVT and LD tests to be able to make direct, withinsubject comparisons between the two procedures for any rat showing deficits under either procedure. These rats were initially trained in the rPVT test, and once their rPVT baseline performances had stabilized, they were trained to perform the LD task. They performed on the LD task until they reached the standard criterion (70% correct responding on the S þ for the discrimination phase) and were then placed on the reversal discrimination until they achieved the same criterion. Rats trained on both procedures performed one task in a 30 min morning session and the other task in a 30 min afternoon session. These rats were randomly assigned to perform the rPVT or LD in the morning session. The method for assigning rats to dose groups was the same as the method previously described for the rPVT rats. Radiation Procedures

Line Orientation Discrimination Test The line orientation discrimination test consisted of a two-stage discrimination-learning task wherein one of two stimuli presented on the screen (S þ) was associated with reinforcement and the other (S–) was not (49, 50). A total of 51 rats (i.e., LD task only rats, see Table 1) were first shaped by the method of successive approximations (51) to touch a large white square on the touch screen to receive a single food pellet for each touch. Once they were reliably responding (i.e., touching inside the white square boundary), the square was progressively reduced in size until rats were responding to a square of the same size for subsequent discrimination testing. Rats were additionally trained to press the square when it appeared randomly in different locations on the screen. Once a rat achieved a 70% or greater accuracy level in this latter stage, it was moved to the discrimination stage of the two-stage LD task. The discrimination stage consisted of the presentation of a pair of stimuli, a horizontal-striped box and vertical-striped box located side-by-side in the middle of the screen, only one stimulus (i.e., S þ ¼ horizontal-striped box) of the pair was associated with reinforcement while the other stimulus (i.e., S– ¼ vertical-striped box) was not. Each trial began with the S þ and S– displayed side-by-side, with a 30 s LH in effect such that an animal had to respond within that time by touching the screen to receive a single food pellet (correct response), otherwise the trial was classified

Animals were exported to Brookhaven National Laboratory (BNL) for radiation exposure 4–5 days prior to the scheduled exposure day. All animals were exposed on the same day and then returned to Johns Hopkins for follow-up testing 4–5 days after exposure. The control group was sham irradiated (i.e., shipped to BNL, sedated and restrained for radiation exposure, but not actually radiated). The remaining rats were irradiated with 150 MeV/n protons generated at the NASA Space Radiation Laboratory (NSRL) facility at BNL. Protons of this energy have a mean range in water of approximately 15.5 cm with an average LET of 0.4 keV/lm. Target exposure levels were 25, 50, 100 and 200 cGy. Actual delivered doses varied by no more than 60.1 cGy relative to each target dose. Dose rates ranged from 55–71 cGy/min except at 25 cGy where the rate was lowered to 30 cGy/min to achieve an even dose distribution across the collimated exposure field. Irradiation involved head-only exposure to minimize systemic responses to exposures that can confound morphological, neurochemical and behavioral testing. For radiation exposures, rats received an intraperitoneal injection (2 ml/kg administered volume) of ketamine (90 mg/kg) and xylazine (5 mg/kg) and were subsequently placed in a ventilated polystyrene holder (2 rats/holder). Each body was shielded by a specially built trilaminar collimator exposure system consisting of Plexiglast, aluminum and polyurethane. The body dose was ,2% of the delivered dose.

ATTENTIONAL DEFICITS FOLLOWING PROTON IRRADIATION

Table 1 shows the number of rats that completed the study under each individual procedure and the combined procedures for each proton exposure dose. All subjects were transported back to Johns Hopkins Medical School within approximately 1 week post-exposure. As all animals were on unrestricted food access while being transported and at BNL, an additional 7–12 days was necessary to slowly reduce the animals’ weights back to normal range for performing the operant tasks. All rPVT rats then performed the rPVT daily (5 days/week) for over an eight month period. All LD rats were tested immediately upon return and then at intervals of approximately every 6 weeks. The rats that were trained on both the rPVT and LD tasks were initially tested on the rPVT task and then placed on the LD task at intervals of approximately 6 weeks. Testing of the basic discrimination and reversal discrimination components of the LD task typically required about 3–5 sessions, after which the animals were returned to the rPVT task. Data Analysis Preirradiation performances of rats trained on the rPVT alone and those trained on the combined LD and rPVT procedures showed no differences in any of the rPVT baseline performance measures, indicating that the addition of the LD procedure did not affect rPVT performance. Similarly, preirradiation performances of rats trained on the LD alone and the combined LD and rPVT did not show any differences in the LD baseline performance measure. Pre- and postirradiation rPVT performances were similar for sham-irradiated controls trained on the rPVT alone or in combination with the LD task (rPVT þ LD). Conversely, pre- and postirradiation LD task performance was similar between sham-irradiated controls trained on the LD task alone or in combination with the rPVT (rPVT þ LD). Given that training on one task did not alter performance of the other task, all data presented here represent results based upon the pooled data (rPVT and rPVT þ LD group for rPVT; LD and rPVT þ LD group for LD task) for each behavioral task. Initial examination of the rPVT data indicated that a number of radiation-exposed animals showed marked deficits in performance after exposure, while other radiation-exposed animals and all control animals did not. Such individual differences were not, however, apparent in LD performance postirradiation. Individual differences were statistically characterized by establishing 95% confidence intervals (CIs) for all animals about each rPVT baseline measure (accuracy, premature responding, false alarm rates, lapse rates and median and mean reaction time), where the baseline was the average performance on the last week prior to radiation. The 95th percentile of all animals’ confidence interals for each measure was then taken as a cutoff point such that an animal’s postirradiation weekly average for each measure had to exceed this cutoff point (i.e., the weekly average for each measure had to be lower than 95% of the weekly control performances of all animals) for that animal to be considered sensitive to the effects of radiation. Two additional criteria were: (1) a radiationexposed animal’s performance had to exceed the maximum number of weeks that any of the control animals showed performance changes beyond the cutoff point, before that animal was considered to show a significant behavioral effect for that measure; and (2) radiationsensitive animals were defined as those showing differences on at least two or more such behavioral measures (e.g., accuracy plus false alarms, etc.). Additionally, to assess the magnitude of radiation-induced deficits over time, an area under the curve (AUC) analysis was conducted for each animal’s rPVT performance for each dependent measure across all days of the post-exposure period (53). With this method, an animal was considered to be radiation-sensitive when its performance fell outside of the range of all control animals’ performances over the same time period. To estimate the likely time course of radiation effects, AUCs were calculated at differing times across the 251 day post-exposure period, starting with day 21 (the first post-exposure day

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when data were available in all animals). AUCs were calculated to extend through approximately 25 day periods post-exposure. Due to variations in daily running schedules (e.g., animals not run on weekends, occasional holidays, etc.), AUCs were calculated from day 21 through day 49, through day 75, through day 97, through day 125, through day 150, through day 175, through day 200, through day 224 and through day 250. Due to multiple comparisons across these time points, the P values were adjusted using Bonferroni type-1 error such that statistical significance was defined as P ¼ 0.00001. To provide an independent verification of the existence of subsets of radiation-sensitive animals, a hierarchical cluster analysis (using Ward’s method) was also employed that groups animals according to a Euclidean distance metric for each behavioral measure, with animals performing more similarly being clustered at less aggregated levels of a hierarchy (54). This method makes no assumptions on the number of clusters that may exist to correspond to a meaningful feature or set of features of the data, and thus provides an independent estimate of the existence of clusters within a data set. This cluster estimate was then employed in a two-step cluster analysis (55) specifically designed to analyze mixed variables measured on different scale levels (as contained in rPVT data), as well as to indicate a variable’s importance for determining a specific cluster. The two-step cluster solutions were conducted across the differing time points (through days 21, 49, 75, 97, 125, 150, 175, 200, 224 and 250) to assess the reliability and repeatability of the resulting groupings. Protein Isolation from Brain Tissue To determine the effects of proton radiation on the integrity of the dopaminergic (DAergic) neurotransmitter system, whole-brain sagittal slices were acquired and protein isolated from rats exposed to 0 (sham), 25, 50 and 100 cGy proton radiation sacrificed at 15 months postirradiation. However, the rats receiving 200 cGy as well as 4 additional sham-irradiated controls were sacrificed at 20 months postirradiation, allowing for completion of further behavioral testing. Radiation-sensitive rats (n ¼ 5 at 200 cGy, n ¼ 4 at 100 cGy, n ¼ 4 at 50 cGy and n ¼ 2 at 25 cGy), radiation-insensitive rats (n ¼ 5 at 200 cGy, n ¼ 3 at 100 cGy, n ¼ 4 at 50 cGy and n ¼ 6 at 25 cGy) and control rats (n ¼ 7 at 15 months and n ¼ 4 at 20 months) were examined to determine the degree that behavioral sensitivity to proton radiation was reflected in subgroup-specific molecular alterations to DAergic protein levels. Whole rat brains were excised after decapitation and immediately stored at 808C. Sixty-micron sagittal slices of one cerebral hemisphere of each rat brain were sectioned with a cryostat using Paxinos and Watson, The Rat Brain in Stereotaxic Coordinates 4th ed. (approximately plate 82). This region of the brain was chosen because it contains several areas important for sustained attention, motor behavior and impulsivity, including the caudate-putamen, nucleus accumbens, ventral tegmental area and substantia nigra. Tissue sections were weighed and placed in a 1.5 ml centrifuge tube. Tissue was homogenized on ice using 3 ml of RIPA buffer containing complete protease inhibitor and phosphatase inhibitors per gram of tissue. Samples were then centrifuged at 12,000g for 10 min at 48C, the pellet was discarded and the lysate transferred to a new microtube. Lysates were again centrifuged at 12,000g for 10 min at 48C. The supernatant from the second centrifugation contains total protein lysate that was used for Western Blotting. Protein concentrations were calculated from each sample using Bio-Rad Protein Assay Dye. Western Blotting to Detect Brain Neurotransmitters Twenty micrograms of total protein from individual rats exposed to the above proton doses were loaded onto 4–12% Bis-Tris gels and electrophoresed for 90 min at 110V in MOPS running buffer. As shown in Fig. 7, protein from individual rats exposed to the same proton dose level or sham irradiation was loaded on a separate gel.

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TABLE 2 Mean rPVT Behavioral Performances (6SD) for Each Proton Dose Group at 175 Days Postirradiation Dose (cGy)

Percent correct

0 25 50 100 200

82.66 81.69 79.95 81.85 79.26

6 6 6 6 6

8.25 15.62 11.78 11.42 14.57

Percent false alarms 9.35 7.10 8.74 7.39 8.82

6 7.69 6 7.52 610.03 6 8.85 6 12.37

Thus each dose level appeared on a gel with several sham-irradiated controls. Proteins were then electrophoretically transferred for 80 min at 30V onto PVDF membrane and subsequently incubated in Ponceau reagent for 10 min to ensure complete transfer of all proteins. After transfer, the membrane was blocked in 5% nonfat dry milk in 13 Tris buffered saline containing 0.1% Tween 20 (TBST). Membranes were incubated with primary antibodies in TBST buffer for 1 h at room temperature. The primary antibodies used were: dopamine transporter (DAT) 1:600; D2, 1:700; and b-actin, 1:2000. Membranes were washed (3 3 10 min) in TBST followed by incubation with secondary antibody at a dilution of 1:2500. After additional washing steps, West Dura chemilluminescent substrate was added to the membranes and they were allowed to incubate for 5 min. Membranes were visualized using a digital ChemiDoc-It imaging system. Densitometry was performed on all Western blots and signal normalized to b-actin, which serves as a housekeeping gene (56). Western Blot Data Analysis After densitometry was calculated, values for individual rats in each proton dose level group characterized as radiation-sensitive or radiation-insensitive were normalized to sham-irradiated controls within each blot, which were set to 1. Given that differences in DAT and D2 protein densities were not apparent for the control rats sacrificed at 15 or 20 months postirradiation (independent sample t tests, all P  0.412), all doses were included in the same analysis. Separate Kruskal-Wallis tests were used to evaluate DAT and D2 protein levels. Mann-Whitney post hoc tests with the false discovery rate alpha correction [FDR (57, 58)] were used to assess specific group differences. When significant protein changes were evident, Spearman correlations were used to determine if DAT or D2 protein densities correlated with the area under the curve data for accuracy and false alarm rate, given that these two measures contributed the most to cluster predictor importance (accuracy . false alarm rate). Correlations were assessed at postirradiation days 125, 150, 175, 200, 224 and 250, since the majority of radiation-sensitive rats were classified as such by day 125. FDR was used to correct P values for multiple correlations. All statistical analyses were completed with IBM SPSS Statistics (version 20.0).

RESULTS

Table 2 shows the effects of proton irradiation on mean performance accuracy, mean percent false alarms, mean percent lapses and median reaction time, respectively, as a function of the proton exposure dose for all animals performing the rPVT procedure. As can be seen, no consistent differences in any of these performance measures were observed when the data were averaged across all animals within each dose group. Figure 1, however, shows examples of the individual differences in rPVT performance accuracy observed after proton irradiation that precipitated the analyses outlined in the Materials and Methods section

Percent lapses 15.45 17.06 17.83 16.97 17.90

6 6 6 6 6

6.79 11.59 8.00 10.37 8.77

Median RT 429.59 497.22 527.40 518.00 552.24

6 6 6 6 6

112.05 123.75 119.26 115.00 148.70

for determining the likely existence of radiation-sensitive and radiation-insensitive subgroups of animals. Shown are graphs of percentage correct scores that were normalized by participant-mean centering [i.e., subtracting the mean of each subject’s preirradiation score from their raw scores and adding back in the preirradiation grand mean of all subjects (59)] for individual animals exposed to 150 MeV/n protons at 200 cGy, 100 cGy, 50 cGy and 25 cGy (closed circles in each graph), relative to the average performance of control animals across the 251 day post-exposure period (open circles). These distinctive decrements in performance were observed within the first month post-exposure with these animals and continued throughout the 251 day postexposure testing period. While there was some variability in the rPVT deficits displayed by rats in each dose group [e.g., spread of closed circles in Fig. 1 (all panels)], there were animals that displayed similar rPVT deficits across all of the doses. In some instances there were no indications of any recovery from the deficits over the testing period (e.g., at 25 cGy, data shown), while in other cases there were indications of partial recovery (e.g., 100 cGy, data shown), and in a few cases recovery back to baseline was observed at around 200–250 days (examples not shown). Additionally, animals showing such decrements in performance also showed an increase in the variability in accuracy of their day-to-day performances, as indicated by the scatter in their performances shown in Fig. 2. This increased variability was verified by an examination of the coefficient of variation (CV, the standard deviation/mean) for animals showing deficits after radiation exposure, relative to controls. Figure 3 shows the average CV for accuracy for animals showing deficits, along with the average CV for all control animals, across the 251 day postexposure period. Variability of performance accuracy clearly increased after radiation exposure, relative to the controls. Based upon the 95% confidence interval analysis, 19 rats were classified as ‘‘radiation-sensitive,’’ and included 7 rats exposed to 200 cGy, 4 rats exposed to 100 cGy, 4 rats exposed to 50 cGy and 4 rats exposed to 25 cGy. Regardless of dose, rats classified as radiation-sensitive consistently maintained rPVT performance levels below those of radiation-insensitive and sham-irradiated control rats throughout the majority of the 251 day post-exposure testing period. Radiation-sensitive rats predominantly showed deficits in accuracy, however, deficits in false

ATTENTIONAL DEFICITS FOLLOWING PROTON IRRADIATION

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FIG. 1. Examples of performance accuracy for animals showing pronounced deficits when exposed to 150 MeV/n protons at 200 cGy, 100 cGy, 50 cGy and 25 cGy. The percent correct scores are shown as a function of days post-exposure, with each dot representing a separate session. Data points to the far left on each graph indicate baseline performances prior to exposure. Shaded areas indicate the range of a 95% confidence interval around the pre-exposure baseline performances of all nonexposed control animals. Closed circles: Animals identified by cluster analysis as being radiation-sensitive; open circles: Average performances of all nonexposed control animals. Solid and dashed lines: Visual fits of data trends to the data, based on centered third order polynomial transforms.

alarm rates, premature responding and lapses in performance were also observed in individual animals throughout the testing period. Median and mean reaction times were for the most part not as profoundly affected in radiationsensitive rats, with only 6 of the 19 radiation-sensitive rats found to show clear elevations (i.e., a slowing) in reaction times, relative to the radiation-insensitive and shamirradiated control rats. Individual differences in area under the curve measures of performance accuracy under the rPVT procedure following proton irradiation are shown in Fig. 3 for animals exposed to protons at 200 cGy, 100 cGy, 50 cGy and 25 cGy. The AUC measure provides a way to quantify the degree to which animals differ from controls in a performance measure over the entire post-exposure testing period. The

relative cumulative percent change in accuracy is shown as a function of days post-exposure, with each line representing a separate animal. The cumulative change is plotted since this measure represents the AUC up to that point in time for each animal, and allows one to easily discern the performances of animals that diverge from the performances of the control animals. As can be seen in Fig. 3, animals classified as radiation-sensitive (see Table 3) showed distinct decrements in performance (thick dotted line) within the first month post-exposure that continued without recovery throughout the 250 day post-exposure testing period, while other irradiated animals did not (solid lines). Performances of control animals (dotted lines) showed no deficits over time. Statistical analysis of the AUC data resulted in 20 rats being classified as radiation-sensitive.

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FIG. 2. Performance variability as measured by daily plots of the coefficient of variation (standard deviation/mean), averaged across animals in the radiation-sensitive and control groups. Closed circles: Animals identified by cluster analysis as being radiation-sensitive; open circles: sham-irradiated controls.

The only difference in classification with the AUC analysis, compared to the 95% CI analysis, occurred in the 200 cGy and 50 cGy groups where one additional rat in each of these dose groups was classified as radiation-sensitive by the AUC analysis, and one rat that had been classified as such with the 95% CI analysis was now classified as radiationinsensitive (differences in the classification of radiationsensitive rats between these analysis methods, as well as the cluster analysis are detailed in Table 3). Cluster analyses of the data indicated that cluster predictor importance mainly came from accuracy, with false alarm rate and lapses contributing to predictor importance less so, and with median RT changes adding little to the overall analysis. Figure 4 shows the results of this analysis followed by a principal components analysis [PCA (60)] of the covariance matrix conducted to validate and visualize the cluster solutions. Figure 4 shows a two-dimensional visual representation of the radiation-sensitive and radiationinsensitive animal clusters based on AUC data through day 175, day 200 and day 224. Such clusters were not evident by day 21, but were clearly present and repeatable at subsequent time periods analyzed throughout the 251 day post-exposure period. With this analysis method, 12 rats were classified as radiation-sensitive and included 5 rats at 200 cGy, 2 rats at both 100 cGy and 50 cGy, and 3 rats at 25cGy. Many of these rats overlapped with rats that were classified as sensitive by the other statistical methods (Table 3). Given the consistency between the methods in grouping many of the same animals together as radiation-sensitive, a

final classification of ‘‘radiation-sensitive’’ was assigned to any rat that was labeled as sensitive by at least two of the three analysis methods. This resulted in n ¼ 7 in the 200 cGy group, n ¼ 4 in the 100 cGy group, n ¼ 5 in the 50 cGy groups and n ¼ 3 in the 25 cGy group. Of these 19 rats, 11 were classified as radiation-sensitive by all three analyses. Table 4 shows a breakdown of the numbers of radiationsensitive vs. radiation-insensitive animals, and indicates that overall, 42% of the animals evidenced radiation sensitivity in terms of a significant neurobehavioral deficit. The percentage of animals showing deficits was distributed across the exposure doses such that no particular dose appears to have been more effective at disrupting rPVT performances (X2 ¼ 1.07, P ¼ 0.30, df ¼ 1). All of the radiation-sensitive animals evidenced radiationinduced decrements in most aspects of rPVT performance (attention, impulsivity and motor function) that began at approximately 50–60 days post-exposure and remained throughout the 251 day testing period. Of the sensitive animals, all showed decrements in performance accuracy. Lapses in attention occurred in 64% of the sensitive subjects, elevations in false alarm rates and premature responding occurred in 45% of the sensitive subjects and elevated median reaction occurred in 27% of the sensitive subjects. Figure 5 shows bar graphs of weekly means for accuracy (top panels) and false alarm rates (bottom panels) (i.e., impulsivity) for baseline performances and the weeks containing AUC days 49, 97, 150, 200 and 250. Data are shown for each measure for both radiation-sensitive and radiation-insensitive animals, plotted as a function of the proton exposure dose, with the grey area depicting the 95% CI for the sham-exposed control performances during these time periods. Radiation-sensitive animals demonstrated deficits that were independent of the irradiated proton dose over the dose range studied (i.e., all doses produced a similar level of behavioral deficits, see Fig. 5). Of the various rPVT behavioral performance measures, accuracy and false alarm rates were most important to cluster membership in the cluster analysis. Notably, radiationinsensitive rats in each dose group had baseline performances similar to those of the sham-irradiated control rats across all post-exposure days. Additional analyses of preexposure baselines for the subsequently identified sensitive and insensitive rats revealed no differences between these two groups for scores of accuracy and false alarms (see Fig. 5, leftmost set of bars in each panel). Additionally, no differences were found for premature responding, lapses, median reaction time, mean reaction, trials completed and food consumed (data not shown). Figure 6 shows the average DAT and D2 receptor protein densities for the 25, 50, 100 and 200 cGy radiation-sensitive rats (black bars), radiation-insensitive (gray bars) and shamirradiated controls (white bars). Radiation-sensitive rats at 100 and 200 cGy displayed significant increases in this protein compared to sham-irradiated controls and radiation-

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265

FIG. 3. Area under the curve plots of accuracy for animals exposed to 150 MeV/n protons at 200 cGy, 100 cGy, 50 cGy and 25 cGy. The relative cumulative percentage change in accuracy is shown as a function of days post-exposure, with each line representing a separate animal. Closed circles: Animals identified by cluster analysis as being radiation-sensitive. Solid lines: Animals identified as being radiation-insensitive; dotted lines: sham-irradiated controls.

insensitive rats at each respective dose (Mann-Whitney post-hoc tests: all P , 0.03). Both radiation-sensitive and insensitive rats at 50 cGy had DAT levels significantly lower than controls (Mann-Whitney post hoc tests: all P , 0.002), but not from each other. DAT protein levels were negatively correlated with accuracy in the 100 and 200 cGy radiation-sensitive rats at all postirradiation days assessed (day 125 rho ¼0.635; day 150 rho ¼0.641; day 175 rho ¼0.630; day 224 rho ¼0.682; day 250 rho ¼0.658; all P  0.007; and day 200 is shown in the lower left panel of Fig. 6). The false alarm rate was positively correlated with DAT density in 100 and 200 cGy radiation-sensitive rats at day 224 only (rho ¼ 0.473; P ¼ 0.030), however, when the outlier in the 100 cGy was removed from the analysis, this correlation was no longer significant (P ¼ 0.107). Even though DAT protein levels were significantly decreased in all rats exposed to 50 cGy, they did not significantly

correlate with area under the curve for changes in either accuracy or false alarm rate. For the D2 receptor densities, the radiation-sensitive rats at 100 cGy had a significantly increased D2 receptor density compared to sham-irradiated controls (Kruskal-Wallis: P ¼ 0.013; Mann-Whitney post hoc test: P , 0.05). No other groups displayed significant differences in D2 receptor protein levels compared to control or radiation-insensitive rats, and no significant correlations were found between accuracy or false alarm rate and D2 density in the 100 cGy radiation-sensitive rats at any of the post-irradiation days tested (Mann-Whitney post hoc tests: all P . 0.072). Data analysis of the LD procedure showed that the cumulative number of trials required to complete the line discrimination reversal learning in control animals varied considerably and encompassed the entire range of performances observed, from best to worst. No additional changes in learning occurred as a function of proton exposure dose.

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TABLE 3 Classification of Radiation-Sensitive Rats by Statistical Methods Subject ID 108 133 139 159 186 190 193 203 178 197 198 202 113 170 176 183 187 122 124 174 191 Total N

Dose (cGy) 200 200 200 200 200 200 200 200 100 100 100 100 50 50 50 50 50 25 25 25 25

95% CIs S S S S S S S S S S S S S S S S S S S 19

AUC S S S S S S S S S S S S S S S S S S S S 20

Cluster analysis S S S S S S S S S S S S 12

Note. S denotes a radiation-sensitive classification by an analysis method.

Finally, for the two animals that were identified by their rPVT performances as being radiation sensitive, their performances in the line discrimination procedure were not affected and fell within the normal range of unexposed control animals. To confirm these findings, an additional cluster analysis was performed with the data to assess

whether subgroups of radiation-sensitive and insensitive animals might also be identified. Unlike the cluster analysis for the rPVT performance data, however, the cluster analysis for the LD data did not correlate with any identifiable variables or reliably categorize control animals or radiation-exposed animals. DISCUSSION

The results clearly show that head-only 25–200 cGy proton irradiation can significantly disrupt neurobehavioral function, as measured by the rPVT procedure, in a readily identifiable subgroup of radiation-sensitive rats. No significant differences were observed between control and radiated animals performing the line discrimination test over the same exposure levels, suggesting that the deficits were specific to the rPVT procedure and not a generalized effect on behavior (e.g., related to a general decline in performance and/or motivation). Thus, there were no foodrelated or motivational changes in performance after irradiation (e.g., no changes were observed in food pellet intake or the total number of trials performed per session). Additionally, as reaction times are known to vary with deprivation level (61), the relatively stable reaction times observed with the current procedure and the low incidence of consistent changes in reaction times after irradiation indicate a minimal effect of irradiation on food consumption or the food-maintained performances of the current study. Further, analyses of pre-exposure baselines of radiationsensitive and radiation-insensitive rats revealed no differences between these two groups for any of the dependent measures examined, suggesting that no obvious preexisting

FIG. 4. Two-dimensional visual representation of the radiation-sensitive and insensitive animal clusters, as determined by a principal components analysis of the covariance matrix of area under the curve data for the independent variables of accuracy, false alarms, and lapses in performance. Cluster analyses are shown for data through 175, 200 and 224 days post-exposure. Prior to principal components analysis, a two-step cluster analysis indicated that cluster predictor importance mainly came from accuracy. False alarm rate and lapses also contributed to predictor importance less so, with median RT changes adding little to the overall analysis. Consequently, principal components analysis was conducted on the area under the curve variables of accuracy, false alarm rate and lapse rate. Clusters were not evident by day 21, but were clearly present at all subsequent time periods.

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ATTENTIONAL DEFICITS FOLLOWING PROTON IRRADIATION

TABLE 4 Distribution of Sensitive and Insensitive Subjects Dose (cGy)

Number of radiation-sensitive rats

Percent

Number of insensitive rats

Percent

Total

200 100 50 25 Total Controls (0)

7 4 5 3 19 0

50% 50% 36% 33% 42% 0%

7 4 9 6 26 11

50% 50% 64% 67% 58% 100%

14 8 14 9 45 11

performance differences contributed to the radiationinduced disruptions. Additionally, the magnitude of the neurobehavioral deficits observed did not appear to be related to the dose level of radiation exposure, suggesting that across the 25–200 cGy exposure levels of this study, equivalent decreases in neurobehavioral function is affected. Finally, only brain tissue from radiation-sensitive rats was found to have altered protein levels associated with changes in DAT that correlated with deficits in rPVT accuracy in 100- and 200-cGy-exposed radiation-sensitive rats, suggesting a possible mechanism for the observed deficits as well as a possible biomarker for identifying radiationsensitive and radiation-resistant individuals, and a potential neurobiological target system for radioprotective interventions. The observed neurobehavioral changes consisted primarily of deficits in accuracy and false alarm rates (‘‘impulsivity’’) that persisted across the 251 day post-exposure testing period, suggesting a profound decrease in the radiationsensitive rats’ abilities to adequately perform the rPVT, with little or no recovery of function apparent for many of the

radiation-sensitive rats. The observed deficits were selective, however, in that changes in the rate of lapses in attention and mean reaction time were not as consistently elevated in the radiation-sensitive rats, relative to the decreases in accuracy and increases in false alarm rates as suggested by the cluster analysis predictor importance (see Results section). In addition to the observed behavioral differences, radiation-sensitive rats exposed to 50, 100 and 200 cGy displayed changes in the levels of the DAT (see Fig. 6). Only 100 cGy radiation-sensitive rats displayed changes in D2 receptor levels. The changes were not, however, consistent across the dose groups, which may be due to the use of whole brain tissue. Interestingly, the significant increases in DAT protein levels in radiation-sensitive rats exposed to 100 and 200 cGy were inversely correlated with their rPVT accuracy levels as early as 125 days postexposure. The lack of a dose-response relationship for the behavioral or biochemical changes observed after proton exposure is not unusual when examining the CNS effects of ionizing radiation. Previous reports have shown dose- or

FIG. 5. Mean weekly accuracy (top panels) and false alarm rates (bottom panels) for radiation-sensitive and insensitive rats for post-exposure week containing days 49, 97, 150, 200 and 250. Accuracy and false alarm rates were the two behavioral measures most important to cluster membership. These graphs demonstrate the lack of a dose response in the rPVT neurobehavioral deficits in the radiation-sensitive rats (black bars) compared to the radiation-insensitive (gray bars) and controls rats (gray horizontal bars representing 95% CI around the mean control percent correct and false alarm rates, respectively). No differences were found in baseline response levels (leftmost bars in each panel; BL ¼ baseline).

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FIG. 6. Average dopamine transporter (DAT; top left panel) and D2 receptor (top right panel) density for the sham-irradiated controls (0 cGy; white bar), 25, 50, 100 and 200 cGy radiation-insensitive (hatched bars) and radiation-sensitive rats (black bars). All samples were normalized to b-actin and are depicted as fold change compared to controls. *Significantly different from control. Alpha ¼ 0.05 level. Bottom left panel: Negative correlation between DAT density and rPVT accuracy (area under the curve) at day 200; closed circles ¼ 100 cGy radiation-sensitive rats; closed triangles ¼ 200 cGy radiation-sensitive rats; open circles ¼ 100 cGy radiation insensitive rats; open triangles ¼ 200 cGy radiation-insensitive rats. Bottom right panel: Individual protein bands; RI ¼ radiation insensitive; RS ¼ radiation sensitive.

ion-specific changes on different behavioral end points and molecular processes in various brain areas, i.e., one dose or ion species can impair some behaviors while leaving others intact, and can decrease molecular processes in one brain area while increasing the same processes in other areas (17, 19, 62). Another possible reason for the lack of a dose effect is that changes in DA-mediated behaviors occur as a function of DA release and are known to have an inverted U-shaped function, where either too little or too much DA release results in behavioral impairments (63, 64). Finally, radiation may damage other neurotransmitter systems that interact with the DA system and thus alter expression levels of the DAT and D2 receptor protein levels indirectly. The observed DA protein changes do, however, represent an interesting preliminary step in beginning to understand the neurobiological underpinnings of radiation sensitivity. The current study suggests that head-only radiation can damage cortical structures and subcortical motor structures, including the frontoparietal attention network important for efficient PVT performance (26, 65) and possibly impair

astronauts’ attentional performances on long-duration missions. Similar to the individual differences described in this article, Basner et al. (32) reported that human PVT performances also differ across individuals when subjects undergo sleep deprivation, suggesting that this additional spaceflight factor could worsen the effects of radiation on attentional performance. While DA-related behavioral deficits are more commonly discussed in terms of attention-deficit hyperactivity disorder [ADHD (66, 67)], human PVT research supports the role of DA in PVT performance in healthy humans, given that alleles resulting in variations in DAT function are associated with individual PVT performance differences (68). These findings stress the importance of understanding DA’s role in attentional performance in healthy subjects, like astronauts and how this system is altered after radiation exposure. The DA system is highly sensitive to damage from oxidative stress and microglial activation, and several types of radiation, including proton radiation, can lead to glial activation and CNS inflammation, altered expression patterns of oxidative

ATTENTIONAL DEFICITS FOLLOWING PROTON IRRADIATION

stress-related genes and changes in the expression and phosphorylation of important cellular signaling protein kinases in the brain, all of which could influence DAergic neurotransmission (16, 17, 19, 62, 69). Finally, the current report is not the only study to describe individual differences in behavioral performance after exposure to protons or HZE particles. Britten2 and colleagues recently reported that a subgroup of 56Fe-exposed rats could not complete the Barnes maze, a hippocampus-dependent test of spatial memory, at 3 months postirradiation. Subsequent proteomic analyses of the hippocampi of these ‘‘bad’’ performers revealed a ‘‘highly disorganized’’ proteomic profile that was not apparent in the ‘‘good’’ performers, i.e., rats that successfully completed the Barnes maze after 56 Fe exposure. Taken together, these results suggest that proton and HZE-induced effects could have behavioral and biological changes that differ by individual, these results suggest the possible existence of biomarkers of vulnerability or resistance to radiation exposure. In summary, the results demonstrate that 150 MeV/n proton irradiations as low as 25 cGy can have pronounced effects on attention and CNS function when examined with an appropriate ground-based animal model that uses longterm neurobehavioral and neurochemical assessments relevant to operational performance in a space radiation environment. Thus, the current animal data and future studies assessing radiation’s effects on rPVT performance can be important for determining acceptable decreases in performance levels after an exposure and developing appropriate mitigation strategies for radiation-induced deficits. Further, the interaction of radiation sensitivity with other space flight factors such as microgravity, fatigue, sleep loss and psychosocial stress will be important areas of future research that may profoundly compound radiation’s effects on neurobehavioral function and the probability of mission success. In this regard, the procedures of the current study may provide an innovative experimental platform for exploring the bases of individual vulnerability to radiationinduced neurobehavioral impairments and evaluating potential prophylactics, countermeasures and treatments for individuals in operational settings characterized by exposure to low-level radiation. ACKNOWLEDGMENT This research was supported by NASA cooperative agreement NCC 9– 58 with the National Space Biomedical Research Institute through NASA NCC 9-58-PF02602 and 9-58-NBPF02802. Received: March 12, 2013; accepted: December 17, 2013; published online: March 10, 2014

2 Britten RA, Birdsall P, Davis L, Lonart G, Drake RR. Executive function is significantly impaired following exposure to low (,20 cGy) of HZE particles. NASA Human Research Performance Investigators’ Workshop, 2012; Houston, TX.

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