NOTE: change in period of performance per July 2013 NSBRI report submission (Ed., 7/12/13)

NOTE: End date change to 5/31/2015 per NSBRI (Ed., 8/23/2012)

Key aims of the study are to determine whether 1) pre-existing individual differences in neurotransmitter function are predictive of the observed differential neurobehavioral susceptibility of individuals to radiation; 2) the observed neurotransmitter changes are restricted to specific neurotransmitter systems and/or brain regions, and 3) differential neurobehavioral susceptibility occurs following exposure to other ion species.

Key Findings:

• Head-only exposure to space radiation particles at mission-relevant doses of protons (p), iron (Fe), or silicon (Si) ¬ions significantly impairs neurobehavioral function (e.g., decreases accuracy, increases impulsivity, increases lapses in attention, slows reaction times).

• Individual rats show a differential susceptibility to radiation, i.e., some rats show an increased sensitivity to radiation while others appear more resistant to radiation effects. Such findings suggest the need for an individualized approach to the measurement and treatment of radiation-induced CNS deficits.

• Radiation-induced differential susceptibility to neurobehavioral dysfunction correlates with increased DA protein levels in radiation-sensitive subjects.

Specific findings from the past year include: Pre-Radiation DA System Status & Neurocognitive Deficits

• Prior to irradiation, the sensitivity of individual animals' to DA compounds was examined via evaluations of the DA agonist d-amphetamine and a DA D2 receptor agonist quinpirole on lever pressing. Post-irradiation tests of the rPVT performances revealed ~30% of the animals were classified as radiation-sensitive (i.e., showing neurocognitive deficits). When the pre-exposure pharmacological data were then re-analyzed as a function of post-exposure radiation sensitivity, rats that eventually showed a sensitivity to radiation exposure also showed an increased sensitivity to d-amphetamine prior to exposure, relative to radiation-insensitive and unexposed control rats, indicating that neurocognitive changes may be linked to individual differences in the pre-radiation DA system status (e.g., increased DA system sensitivity may be predictive of subsequent radiation sensitivity).

Dopaminergic Modulation of Radiation-Induced Neurocognitive Deficits

• Radiation-induced deficits in rPVT performances are mitigated by injections of the DA agonist d-amphetamine. Following proton irradiation, radiation-sensitive animals show decreased performance accuracy and slowed reaction times, while sham-irradiated controls and radiation-insensitive animals do not. Administrations of d-amphetamine (0.56, 1.0, 1.8, or 3.2 mg/kg) produce dose-dependent recovery of both accuracy and reaction time speed in radiation-sensitive animals. Additional pharmacological evaluations of the norepinephrine reuptake inhibitor atomoxetine showed no differential effects on rPVT performance on radiation-sensitive vs. insensitive rats. Taken together, these data provide additional evidence of the specific involvement of the DA system in radiation-induced neurobehavioral deficits.

Changes in Dopaminergic Modulation following Radiation

• Drug-induced yawning is a sensitive metric for determining subtle changes in the DA system. Administration of a DA D2/D3 receptor agonist results in a predictable pattern of drug-induced yawning in which yawning frequency first increases as the drug dose is increased, and then decreases at successively higher doses. This rising and falling pattern results from activation of the yawning reflex by D3 receptors on the ascending limb, and by inhibition of yawning by D2 receptors on the descending limb. Following 100 cGy proton exposures, differential shifts in the ascending limbs of these curves have been observed: 1) yawning is reduced in radiation sensitive rats and is unchanged following administration of a D2 antagonist, suggesting continued D2 receptor hypersensitivity and possibly increased D2 receptor levels; 2) yawning is greatest in radiation-insensitive rats and increases dose-dependently following administration of a D2 antagonist, which suggests a faster decrease in D2 hypersensitivity, in addition to altered D3 receptor levels.

In sum, the results suggest altered DA signaling in radiation-sensitive rats. A new publication describes the effects of proton irradiation on rodents' performance of an automated intra-dimensional set-shifting task. Results showed decreased responding and elevated numbers of omitted trials during the first two performance stages; when tested on an added social recognition memory test, the same animals showed no significant memory effects.

Plans for the Coming Year: This was the last year of the project.

Specific findings from the past year include: Prior to irradiation, the sensitivity of individual animals' to DA compounds was examined via evaluations of the DA agonist d-amphetamine and a DA D2 receptor agonist quinpirole on lever pressing. Post-irradiation tests of the rPVT performances revealed ~30% of the animals were classified as radiation-sensitive (i.e., showing neurocognitive deficits). When the pre-exposure pharmacological data were then re-analyzed as a function of post-exposure radiation sensitivity, rats that eventually showed sensitivity to radiation exposure also showed an increased sensitivity to the DA agonist prior to exposure, indicating that neurocognitive changes may be linked to increased DA system sensitivity prior to radiation.

Dopaminergic Modulation of Radiation-Induced Neurocognitive Deficits: Radiation-induced deficits in rPVT performances are mitigated by injections of the DA agonist d-amphetamine. Radiation-sensitive animals show decreases in performance accuracy and slowed reaction times; administration of d-amphetamine, however, produces dose-dependent recovery of both performance accuracy and reaction time speed in radiation-sensitive animals. The norepinephrine reuptake inhibitor atomoxetine shows no differential effects on rPVT performance on radiation-sensitive vs. insensitive rats, which suggests the specific involvement of the DA system in these neurobehavioral deficits.

Changes in Dopaminergic Modulation following Radiation: Drug-induced yawning is a sensitive metric for determining subtle changes in the DA system. Administration of a DA agonist produces predictable rising and falling pattern of drug-induced yawning in which yawning first increases as the drug dose is increased, and then decreases at successively higher doses. This pattern results from activation of yawning by D3 receptors on the ascending limb, and by inhibition of yawning by D2 receptors on the descending limb. Proton exposures produce differential shifts in the ascending limbs of these curves: 1) yawning is reduced in radiation sensitive rats and is unchanged following administration of a D2 receptor antagonist, which suggests continued D2 receptor hypersensitivity and possibly increased D2 receptor levels; 2) yawning is greatest in the radiation insensitive rats and dose-dependently increases following administration of a D2 receptor antagonist, which suggests a faster decrease in D2 hypersensitivity in addition to altered D3 receptor levels and/or function. In sum, the results suggest altered DA signaling in radiation-sensitive rats.

NOTE: change in period of performance per July 2013 NSBRI report submission (Ed., 7/12/13)

NOTE: End date change to 5/31/2015 per NSBRI (Ed., 8/23/2012)

Cognitive neurobehavioral functions relevant to astronaut mission performance effectiveness are being assessed with a rodent analog of the human Psychomotor Vigilance Test (PVT) currently used in space analog environments and by astronauts aboard ISS, which includes assessments of general motor function and speed, vigilance, inhibitory control ('impulsivity'), timing, motivation, and basic sensory function. Animals trained on the rodent version of the PVT (the rPVT) are subsequently exposed to protons and high-energy particle radiation and then tested for up to 12 months post-exposure to assess potential short- and long-term performance deficits. Likely mechanisms of damage to the CNS following radiation exposure are examined via pre-radiation behavioral pharmacology studies as well as post-radiation behavioral pharmacology studies and neurochemical assessments of CNS proteins relevant to neurotransmitter function and inflammation.

Key aims of the study are to determine 1) whether pre-existing individual differences in neurotransmitter function may be predictive of the observed differential neurobehavioral susceptibility of individuals following proton radiation; 2) whether the observed neurotransmitter changes are restricted to specific brain regions; and 3) whether differential neurobehavioral susceptibility occurs following exposure to other ion species.

Key Findings: Results from the project have demonstrated that head-only exposure to space radiation (protons, 56Fe, 28Si) significantly impairs neurobehavioral function (e.g., decrease accuracy, increase impulsivity, increase lapses in attention) and slows motor function. These findings support the success of the rPVT as a rodent model for studying the risks of living in the space radiation environment due to changes in neurobehavioral function.

Specific findings from the past year include:

1. Pre-exposure evaluations of a DA agonist and antagonist on FR/FI performance were completed. Following radiation, retests on the rPVT performances revealed ~30% of the animals to be classified as radiation-sensitive. Post-exposure pharmacological challenges with a DA agonist and antagonist are underway to determine whether radiation-induced changes in the DA system are linked to pre-radiation DA system changes (i.e., whether pre-radiation DA system sensitivities may be predictive of subsequent radiation sensitivity).

2. Drug-induced yawning is being used as a sensitive metric for determining subtle changes in D2 and D3 activity in rodents. A rising and falling pattern of DA-induced yawning in rodents is induced by activation of the D3 receptor on the ascending limb, and by inhibition by the D2 receptor on the descending limb. This differential modulation by D3 and D2 antagonists results in D3-preferring antagonists producing selective rightward shifts of the ascending limb, and D2-preferring antagonists producing selective shifts of the descending limb. Following 25-100 cGy proton exposures, differential shifts in the ascending limbs of these curves have been observed, suggesting that D3 receptor activity and/or tissue levels are altered by radiation.

3. A study (part of Catherine Davis' NSBRI Postdoctoral Fellowship) determined the degree to which radiation-induced deficits in neurobehavioral function differ as a function of changes in cytokine protein expression. Brain tissue of both F344 and LEW rPVT-trained rats were found to have differential changes in frontal cortex cytokine levels; several neurotrophic and putative pro-cognitive cytokines were significantly elevated in the LEW rats, which could underlie the lack of radiation-induced rPVT deficits in this strain. Western blot analyses of several different proteins important for dopamine neurotransmission (e.g., dopamine transporter, D2 receptor, tyrosine hydroxylase) and cell survival (e.g., Akt, p-Akt, CREB), in addition to various cytokines (e.g., TNF-a, Il-1a, Il-6, GM-CSF, CNTF, VEGF) in the frontal and parietal cortices of the F344 and LEW rats were found to be differentially altered following proton exposure.

4. Two new publications have appeared that demonstrate 1) Exposure to head-only proton irradiation differentially disrupts rPVT performance in a subgroup of radiation-sensitive animals, and that these deficits are correlated with changes in the levels of the dopamine transporter and the D2 receptor in this subgroup; and 2) following proton irradiation, rats performing an automated intra-dimensional set shifting task respond less and have elevated numbers of omitted trials during the first two performance stages, but show no effects of radiation on social recognition memory.

Plans for the Coming Year: Plans include completing 1) behavioral pharmacology studies to determine the degree to which pre-existing individual differences in neurotransmitter function may be predictive of the observed differential neurobehavioral susceptibility of individuals following radiation, 2) neurotransmitter protein level studies to determine the degree to which the observed neurotransmitter changes are restricted to specific brain regions, and 3) continued support of Dr. Catherine Davis' NSBRI Postdoctoral Fellowship studies designed to assess neurochemical changes in the brain following radiation.

1. Neurotransmitter studies are assessing levels of neurotransmitter protein, terminal degradation (via changes in related protein levels), and inflammatory proteins commonly active following brain trauma. Tissue has been collected at 1 and 5 months post-radiation, and neurochemical cytokine analyses are ongoing.

2. Behavioral pharmacology studies are examining radiation effects on the integrity of the DA system in trained rats via comparison of pre- and post-radiation assessments of their sensitivity to neurotransmitter receptor indirect agonists, agonists, and antagonists via 3 procedures: the rPVT procedure, a mixed FR-30/FI-2 min operant schedule, and a drug-induced yawning procedure.

A. Pre-exposure evaluations of a DA agonist and antagonist on FR/FI performance were completed and the animals subsequently irradiated. Post-exposure challenges with a these same drugs are underway to assess whether radiation-induced changes in the DA system are linked to pre-radiation DA system changes (i.e., whether pre-radiation DA system sensitivities may be predictive of subsequent radiation sensitivity).

B. Drug-induced yawning is a sensitive metric for determining subtle changes in D2 and D3 activity in rodents. A rising and falling pattern of DA-induced yawning in rodents is induced by activation of the D3 receptor on the ascending limb, while inhibition of yawning is observed at higher doses (descending limb) via concomitant activation of the D2 receptor. Following 25-100 cGy proton exposures, differential shifts in these curves have been observed, suggesting that activity of these receptors is altered following exposure.

3. A study (part of Catherine Davis' NSBRI Postdoctoral Fellowship) determined the degree to which radiation-induced deficits in neurobehavioral function differ as a function of changes in cytokine protein expression. Brain tissue of both F344 and LEW rPVT-trained rats were found to have differential changes in frontal cortex cytokine levels; several neurotrophic cytokines were significantly elevated in the LEW rats, which could underlie the lack of radiation-induced rPVT deficits in this strain. Many of these same cytokines were significantly decreased in the irradiated F344 rats, the strain displaying rPVT deficits following radiation. Analyses of several different proteins important for dopamine neurotransmission (e.g., dopamine transporter, D2 receptor, tyrosine hydroxylase) and cell survival (e.g., Akt, p-Akt, CREB), in addition to various cytokines (e.g., TNF-a, Il-1a, Il-6, GM-CSF, CNTF, VEGF) in the frontal and parietal cortices of the F344 and LEW rats have been found to be differentially altered following proton exposure.

NOTE: change in period of performance per July 2013 NSBRI report submission (Ed., 7/12/13)

NOTE: End date change to 5/31/2015 per NSBRI (Ed., 8/23/2012)

Cognitive neurobehavioral functions relevant to astronaut mission performance effectiveness are being assessed with a rodent analog of the human Psychomotor Vigilance Test (PVT) currently used in space analog environments and by astronauts aboard ISS, which includes assessments of general motor function and speed, vigilance, inhibitory control ('impulsivity'), timing, motivation, and basic sensory function. Animals trained on the rodent version of the PVT (the rPVT) are subsequently exposed to protons and high-energy particle radiation and then tested for up to 12 months post-exposure to assess potential short- and long-term performance deficits. Likely mechanisms of damage to the CNS following radiation exposure are examined via pre-radiation behavioral pharmacology studies as well as post-radiation behavioral pharmacology studies and neurochemical assessments of CNS proteins relevant to neurotransmitter function and inflammation.

Key aims of the study are to determine 1) whether pre-existing individual differences in neurotransmitter function may be predictive of the observed differential neurobehavioral susceptibility of individuals following proton radiation; 2) whether the observed neurotransmitter changes are restricted to specific brain regions, and 3) whether differential neurobehavioral susceptibility occurs following exposure to other ion species.

Key Findings: Results from the project have demonstrated the reliability and validity of the neurobehavioral procedures in detecting behavioral changes following radiation over extended intervals following radiation exposure. Head-only exposure to radiation (protons, 56Fe, 28Si) has been shown to significantly impair neurobehavioral function (e.g., decrease accuracy, increase impulsivity, increase lapses in attention) and slow motor function. These findings support the success of the rPVT as a rodent model for studying the risks of living in the space radiation environment due to changes in neurobehavioral function.

Specific findings from the past year include: 1. Demonstrating that exposure to 28Si ions produces highly specific effects on vigilance that are similar to the effects previously observed with both proton and 56Fe exposures, and include an individual differences effect in that only a subset of irradiated animals show neurobehavioral deficits (i.e., are radiation sensitive ). In contrast to both proton and 56Fe radiation, 28Si radiation produced deficits in only about 9% of the exposed animals, suggesting a lower level of CNS-related dysfunction associated with 28Si radiation exposure.

2. Determining the degree to which behavioral responses to dopaminergic drugs differ as a function of radiation sensitivity. Radiation-sensitive and –insensitive rats were pharmacologically challenged with quinpirole (a D2 receptor agonist) and amisulpride (D2 receptor antagonist), with their behavior assessed via rate-decreasing effects of the drugs on fixed-ratio responding (a widely-used in vivo method for determining possible neurochemical differences). Results indicate 1) a likely increase in D2 receptor availability in insensitive rats, suggestive of a possible protective mechanism following radiation exposure, 2) that radiation-sensitive rats likely have decreased DA neurotransmission, and 3) that amisulpride administration may be a possible mitigation strategy to alleviate radiation-induced deficits on rPVT performance in sensitive rats.

3. Determining the radioprotective effects of a 10% flax seed diet for mitigating proton-induced neurobehavioral deficits (part of Dr. Catherine Davis' NSBRI Postdoctoral Fellowship PF02602). While the flax seed did not affect the proportion of animals developing neurocognitive deficits, there was a trend indicating that the animals receiving flax seed may show signs of early recovery from these deficits, a trend not evident in the irradiated rats not on flax seed.

4. Determining the degree to which radiation-induced deficits in neurobehavioral function differ as a function of basal dopaminergic function (supported by this grant and part of Dr. Catherine Davis's NSBRI Postdoctoral Fellowship PF02602). Brain tissue of both F344 and LEW rats displaying long-term deficits in rPVT performance were examined to determine if chronic inflammatory changes were evident. Significant changes in frontal cortex cytokine levels were found only in those F344 rats displaying a radiation-induced behavioral deficit in the rPVT, suggesting a chronic inflammatory state in the brain could damage numerous areas important for sustained attention and impulsive behavior.

Plans for the Coming Year: Plans include 1) behavioral pharmacology studies to determine the degree to which pre-existing individual differences in neurotransmitter function may be predictive of the observed differential neurobehavioral susceptibility of individuals following radiation, 2) neurotransmitter protein level studies to determine the degree to which the observed neurotransmitter changes are restricted to specific brain regions, and 3) continued support of Dr. Catherine Davis' NSBRI Postdoctoral Fellowship studies designed to assess neurochemical changes in the brain following radiation.

1. One study (N=60) examined the effects of 28Si radiation (0, 10, 25, and 50 cGy, 300 MeV/n) on rPVT performance, and showed that 28Si ions produce highly specific effects on vigilance that are similar to the effects previously observed with both proton and 56Fe radiation. 28Si also produces an individual differences effect in that only a subset of irradiated animals show neurobehavioral deficits, with these deficits also being independent of dose. 28Si radiation produced deficits in only about 9% of the exposed animals, suggesting a lower level of CNS-related dysfunction associated with exposure to this ion species.

2. A second study (N=50, part of Dr. Catherine Davis' NSBRI Postdoctoral Fellowship) determined the radioprotective effects of a 10% flax seed diet for mitigating proton-induced neurobehavioral deficits. While the flax seed did not affect the proportion of animals developing neurocognitive deficits, there was a trend indicating that the animals receiving flax seed may show signs of early recovery from these deficits, a trend not evident in the irradiated rats not on flax seed.

3. A third study determined the degree to which behavioral responses to dopaminergic drugs differ as a function of radiation sensitivity on the rPVT. Radiation-sensitive and -insensitive rats were pharmacologically challenged with quinpirole (a D2 receptor agonist) and amisulpride (D2 receptor antagonist), with their behavior assessed via rate-decreasing effects of the drugs on fixed-ratio responding (a widely-used in vivo method for determining possible neurochemical differences). Results indicate 1) a likely increase in D2 receptor availability in insensitive rats, suggestive of a possible protective mechanism following radiation exposure, 2) that radiation-sensitive rats likely have decreased DA neurotransmission, and 3) that amisulpride administration may be a possible mitigation strategy to alleviate radiation-induced deficits on rPVT performance in sensitive rats.

4. A fourth study (part of Catherine Davis' NSBRI Postdoctoral Fellowship) determined the degree to which radiation-induced deficits in neurobehavioral function differ as a function of basal dopaminergic function. Brain tissue of both F344 and LEW rats displaying long-term deficits in rPVT performance were examined to determine if chronic inflammatory changes were evident. Significant changes in frontal cortex cytokine levels were found only in those F344 rats displaying a radiation-induced behavioral deficit in the rPVT, suggesting a chronic inflammatory state in the brain could damage numerous areas important for sustained attention and impulsive behavior.

To assess the likelihood of space radiation producing long-term functional changes in the CNS, neurobehavioral functions will be measured in rodents via animal tests analogous to human 'vigilance' tests in humans. Cognitive neurobehavioral functions relevant to astronaut mission performance effectiveness will be assessed with a rodent analog of the Psychomotor Vigilance Test (PVT) currently used in space analog environments and by astronauts aboard ISS. Neurobehavioral functions to be examined include assessments of general motor function and speed, vigilance, inhibitory control ('impulsivity'), timing, motivation, and basic sensory function. Groups of animals will be trained on the rodent version of the PVT, following which they will be exposed to radiation and then re-tested periodically for up to 18 months post-exposure to assess potential long-term performance deficits. Likely mechanisms of damage to the CNS following radiation exposure will be examined via pre-radiation behavioral pharmacology studies as well as post-radiation behavioral pharmacology studies and neurochemical assessments (Western blots) of proteins relevant to neurotransmitter function and inflammation.