Fractionated Proton Irradiation Does Not Impair Hippocampal-Dependent Short-Term or Spatial Memory in Female Mice

Abstract

The environment outside the Earth's protective magnetosphere is a much more threatening and complex space environment. The dominant causes for radiation exposure, solar particle events and galactic cosmic rays, contain high-energy protons. In space, astronauts need healthy and highly functioning cognitive abilities, of which the hippocampus plays a key role. Therefore, understanding the effects of ¹H exposure on hippocampal-dependent cognition is vital for developing mitigative strategies and protective countermeasures for future missions. To investigate these effects, we subjected 6-month-old female CD1 mice to 0.75 Gy fractionated ¹H (250 MeV) whole-body irradiation at the NASA Space Radiation Laboratory. The cognitive performance of the mice was tested 3 months after irradiation using Y-maze and Morris water maze tests. Both sham-irradiated and ¹H-irradiated mice significantly preferred exploration of the novel arm compared to the familiar and start arms, indicating intact spatial and short-term memory. Both groups statistically spent more time in the target quadrant, indicating spatial memory retention. There were no significant differences in neurogenic and gliogenic cell counts after irradiation. In addition, proteomic analysis revealed no significant upregulation or downregulation of proteins related to behavior, neurological disease, or neural morphology. Our data suggests ¹H exposure does not impair hippocampal-dependent spatial or short-term memory in female mice.

Keywords: behavior; hippocampus; radiation; space.

Scheme 1

Timeline of experimental design for fractionated irradiation, behaviorial testing and euthanasia.

Figure 1

Y-maze. Both sham-irradiated (A) and ¹H-irradiated (B) mice significantly preferred exploration of the novel arm compared to the familiar and start arms ** p < 0.01.

Figure 2

Y-maze discrimination ratio. Sham- and ¹H-irradiated animals displayed a positive discrimination ratio. There was no difference in discrimination ratios preferred to explore the novel arm.

Figure 3

Morris water maze swim velocity. There was no significant treatment-by-day interaction for velocity. Hidden indicates the platform was not visible during the training trials.

Figure 4

Morris water maze latency. There were no significant differences in treatment-by-day interactions for latency. Hidden indicates the platform was not visible during the training trials.

Figure 5

Morris water maze distance moved. There was a significant difference in time (F_{(2.9, 52.20)} = 25.00, p < 0.001). Hidden indicates the platform was not visible during the training trials.

Figure 6

Morris water maze probe trials on day 5. Both the sham- and fractionated ¹H-irradiated mice spent more time in the target quadrant compared to the right, opposite and left quadrants. * p < 0.001.

Figure 7

(A) Newly born cells. There was a significant difference in the numbers of BrdU⁺ cells in the fractionated ¹H-irradiated mice compared to sham-irradiated mice (* p < 0.05). (B) Newly born neurons (NeuN⁺/BrdU⁺). There were no significant differences in the numbers of cells/mm² between groups (p = 0.40) ¹H-irradiated mice (N = 6) compared to sham-irradiated mice (N = 6). (C) Newly born astrocytes (GFAP^+/BrdU⁺). There were no significant differences in the numbers of newly born astrocytes between groups (p = 0.69). (D) Activated microglia (CD68 only). There were no significant differences in the total numbers of activated microglia between groups (p = 0.07). (E) Newly born activated microglia (BrdU⁺/CD68⁺). There were no significant differences in the numbers of newly born activated microglia between groups (p = 0.15).

Figure 8

Graphic representation of mouse hippocampus protein network 1, identified by Ingenuity Pathway Analysis (IPA) as being affected by both sham irradiation and ¹H irradiation. Functions associated with the network include developmental disorder, hereditary disorder, and metabolic disease. The network is overlayed with the disease and function tool to display the 3 key molecules involved with behavior. The node color indicates expression value, and color intensity indicates degree of upregulation (red) or downregulation (green). Gray nodes are dataset molecules that were not significantly expressed and therefore did not pass the IPA analysis cutoff. Uncolored nodes were not part of our dataset but were incorporated into the pathway based on evidence stored in the Ingenuity Knowledge Base. Known direct and indirect interactions between network proteins, as well as the direction of the interaction, are indicated by arrows or blocked lines.

Figure 9

Visual diagram of the mitochondrial dysfunction canonical pathway identified by Ingenuity Pathway Analysis (IPA) as being associated with both the sham-irradiated group and the ¹H-irradiated group. The diagram provides an image of how the pathway is structured, where the molecules from the dataset are located, and a prediction of molecule activity. The nodes outlined in magenta are the molecules found in the dataset. The node color indicates expression value, and color intensity indicates degree of upregulation (red) or downregulation (green). Gray nodes are dataset molecules that were not significantly expressed and therefore did not pass the IPA analysis cutoff. Uncolored nodes were not part of our dataset but were incorporated into the pathway based on evidence stored in the Ingenuity Knowledge Base. Blue indicates a prediction of inhibited activity. Solid lines indicate a direct interaction between molecules and the predicted relationship between the molecules. Grey arrows indicate the effect is not predicted. The direction of the interaction is represented by arrows. For signaling pathways, an arrow pointing from one molecule to another indicates the cause of activation.