Alteration of Interneuron Immunoreactivity and Autophagic Activity in Rat Hippocampus after Single High-Dose Whole-Brain Irradiation

Abstract

The effects of high dose gamma radiation on brain tissue are poorly understood, with both limited and major changes reported. The present study compared the effects of gamma irradiation on the expression of interneuron markers within the hippocampal cornu ammonis 1 (CA1) region with expression in control matched rats. This area was chosen for study because of its well-characterized circuitry. Male Sprague-Dawley rats were exposed to 60 Gy of whole brain gamma radiation and after 24 or 48 hours, the brains were removed, fixed and sectioned to quantitate expression of parvalbumin (PV), calbindin-D28K (CB), reelin, neuropeptide-Y (NPY), and somatostatin. All of these markers increased in expression over the first 48 hours, except NPY, which decreased. This provides novel information on changes in gene expression in the hippocampal interneurons following radiation. Staining for Beclin 1, a marker of autophagy, increased most strongly in the subgranular zone (SGZ) of the dentate gyrus (DG). Overall, the results are consistent with the hypothesis that increased intracellular calcium follows irradiation, leading to an increased expression of calcium binding proteins. Increased autophagy occurs in the neurogenic zone of the dentate hilus, consistent with reduced effective neurogenesis after irradiation.

Keywords: autophagy; calbindin; hippocampus; interneurons; neuropeptide y; parvalbumin; reelin; whole-brain radiation.

Figure 1. Diagram of the hippocampus showing subregions

CA1: cornu ammonis region 1; CA2: cornu ammonis region 2; CA3: cornu ammonis region 3; DG: dentate gyrus; fim: fimbria pathway; mf: mossy fiber pathway; sc: Schaffer collateral pathway; alv: alveus; pp: perforant pathway axons.

Figure 2. Parvalbumin (PV) increases in hippocampal CA1 area at 24-hour and 48-hour recovery after a single dose of 60 Gy gamma radiation

(A) Representative images showing PV staining (red color) in the hippocampus from sham, and after 24-hours and 48-hours recovery following irradiation. (B) Higher power views of hippocampal CA1 (boxes in A) indicate where the PV-positive neurons were counted. (C) Quantification of PV-positive neurons in hippocampal cornu ammonis 1 (CA1) area shown at higher magnification in panel B; N=6 rats/group x 5 slices/animal, P<0.01 compared to sham (*) or to 24 hours (#). (D) Clusters of PV-positive neurons were observed in the 48-hour recovery group but not in sham; black arrows indicate the CA1 layers. Scale bars: A, 400 µM; B, 200 µM; D, 25 µM.

Figure 3. Calbindin D-28K (CB) increases in the hippocampal CA1 area 24 hours and 48 hours after a single dose of 60 Gy gamma radiation

(A) Representative images showing CB staining (red color) in the hippocampus from sham, and rat brains 24 hours and 48 hours after irradiation. (B) Higher power views of hippocampal CA1 (boxes in A) indicate where the CB-positive neurons were counted; arrows show the hippocampal CA1 pyramidal layer. (C) Quantification of CB-positive neurons in the hippocampal CA1 area shown in B; N=6 rats/group x 5 slices/animal, P<0.01 compared to sham (*) or to 24 hours (#); Black arrows indicate CA1 layers. Scale bars: A, 400 µM; B, 120 µM.

Figure 4. Reelin-positive cells increase in the hippocampal CA1 area 24-hours and 48-hours recovery after a single dose of 60 Gy gamma radiation

(A) Representative images showing reelin staining (red color) in hippocampus from sham, and after 24-hours and 48-hours recovery following irradiation; cell nuclei are stained with 4’6-diamidino-2-phenylindol (DAPI) (blue). (B) Higher power views of hippocampal CA1 (boxes in A) indicate the areas in which reelin-positive neurons were counted. (C) Quantification of reelin-positive neurons in CA1 within the area shown in panel B; N=6 rats/group x 5 slices/animal, P<0.01 compared to sham (*); black arrows indicate CA1 layers. Scale bars: A, 400 µM; B, 200 µM.

Figure 5. Neuropeptide Y (NPY) decreases in the hippocampal CA1 at 24 hours and 48 hours after a single dose of 60 Gy gamma radiation

(A) Representative images showing NPY staining (red color) in the hippocampal CA1 area from sham, and brain harvested 24 hours and 48 hours after irradiation; cell nuclei are stained with DAPI (blue). (B) Quantification of NPY-positive neurons in the hippocampal CA1 area illustrated in A; N=6 rats/group x 5 slices/animal, P<0.01 compared to sham (*) or to 24 hours (#); black arrows indicate CA1 layers. Scale bar: 200 µM.

Figure 6. Changes of beclin 1 staining in hippocampal CA1 and DG areas 24 hours and 48 hours after a single dose of 60 Gy gamma radiation

(A) Representative images showing beclin 1 staining in the hippocampus from sham, and brains harvested 24 hours and 48 hours after irradiation. (B) Quantification of beclin-1 immunoreactivity in hippocampal CA1 and dentate gyrus (DG) areas showed in A; average intensity was measured in 6 rats/group x 3 slices/animal, P<0.01 compared to sham (*); black arrows indicate CA1 layers. Scale bar: 400 µM.