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. 2009 Jan;171(1):41-52.
doi: 10.1667/RR1361.1.

Alterations in Glutamate Uptake in NT2-derived Neurons and Astrocytes After Exposure to Gamma Radiation

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Free PMC article

Alterations in Glutamate Uptake in NT2-derived Neurons and Astrocytes After Exposure to Gamma Radiation

Martha C Sanchez et al. Radiat Res. .
Free PMC article

Abstract

Currently, the cellular and molecular mechanisms that underlie radiation-induced damage in the CNS are unclear. The present study began investigations of the underlying mechanism(s) for radiation-induced neurotoxicity by characterizing glutamate transport expression and function in neurons and astrocytes after exposure to gamma rays. NTera2-derived neurons and astrocytes, isolated as pure cultures, were exposed to doses of 10 cGy, 50 cGy and 2 Gy gamma rays, and transporter expression and function were assessed 3 h, 2 days and 7 days after exposure. In neurons, at 7 days after exposure, a significant increase was detected in EAAT3 after 50 cGy (P < 0.05) and a dose-dependent increase in GLT-1 expression was seen between doses of 10 and 50 cGy (P < 0.05). Functional assays of glutamate uptake revealed that neurons and astrocytes respond in a reciprocal manner after irradiation. Neurons responded to radiation exposure by increased glutamate uptake, an effect still evident at our last time (7 days) after exposure (P < 0.05). The astrocyte response to gamma radiation was an initial decrease in uptake followed by recovery to baseline levels at 2 days after exposure (P < 0.05). The observations made in this study demonstrate that neurons and astrocytes, while part of the same multifunctional unit, have distinct functional and reciprocal responses. The response in neurons appears to indicate a protracted response with potential long-term effects after irradiation.

Figures

FIG. 1
FIG. 1. NT2/N and NT2/A cell morphology
Phase-contrast image of NT2-derived neurons and astrocytes. Panels a and b: NT2/N cells display classic neuron morphology with soma and extended processes. Panels c and d: NT2/A cells exhibit morphology characteristic of protoplasmic astrocytes. Original magnification 10× in panels a and c and 20× in panels b and d.
FIG. 2
FIG. 2. Differentiated NT2 cells immunolabeled with markers of mature neuron and astrocyte phenotypes
Panel a: NT2-derived neurons labeled with the neuronal nuclear marker NeuN (green). Panel b: Astrocytes labeled with GFAP (green). In both images the nuclei are counterstained red with propidium iodide. Original magnification 40×.
FIG. 3
FIG. 3. Immunolabeling of glutamate transporters in astrocytes
GLAST (panel a), GLT-1 (panel b), EAAT3 (panel c) and EAAT4 (panel d) were secondarily labeled green and the nuclei were counterstained red with PI. The images are representative of the diffuse immunolabeling pattern observed in these cultures. Original magnification 40×.
FIG. 4
FIG. 4. Western blot of GLT1 glutamate transporter expression in astrocytes and neurons
The blot shows glutamate transporter EAAT-2 (GLT1) protein expression 2 days after exposure to γ rays at the doses indicated. RBL, rat brain lysate.
FIG. 5
FIG. 5. Functional assessment of glutamate transporters in isolated neuron cultures after γ irradiation
Functional activity was assessed directly by measuring the uptake of [3H]glutamate in NT/N cultures 3 h, 2 days, and 7 days after irradiation. The time course for uptake ranged from 0 to 90 min after initial addition of [3H]glutamate to cultures. Data are the average values of six biological replicates ± SD.
FIG. 6
FIG. 6. Functional assessment of glutamate transporters in isolated astrocyte cultures after γ irradiation
Functional activity was assessed directly by measuring the uptake of [3H]glutamate in NT/A cultures 3 h, 2 days and 7 days after irradiation. The time course for uptake ranged from 0 to 90 min after initial addition of [3H]glutamate to cultures. Data are the average values of six biological replicates ± SD.
FIG. 7
FIG. 7. Functional assessment of glutamate transporters in normal fetal astrocytes after γ irradiation
Functional activity was assessed directly by measuring the uptake of [3H]glutamate in normal fetal astrocyte cultures 3 h and 2 days after irradiation. The time course for uptake ranged from 0 to 90 min after initial addition of [3H]glutamate to the cultures. Data are the average values of three biological replicates ± SD.
FIG. 8
FIG. 8. Cellular viability in neurons and astrocytes after irradiation
Percentage cell loss was assessed 3 h, 2 days and 7 days after irradiation. Data are the average values of six biological replicates ± SD.

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