Neuroprotective role of dopamine against hippocampal cell death

Abstract

Glutamate excitotoxicity plays a key role in the induction of neuronal cell death occurring in many neuropathologies, including epilepsy. Systemic administration of the glutamatergic agonist kainic acid (KA) is a well characterized model to study epilepsy-induced brain damage. KA-evoked seizures in mice result in hippocampal cell death, with the exception of some strains that are resistant to KA excitotoxicity. Little is known about the factors that prevent epilepsy-related neurodegeneration. Here we show that dopamine has such a function through the activation of the D2 receptor (D2R). D2R gene inactivation confers susceptibility to KA excitotoxicity in two mouse strains known to be resistant to KA-induced neurodegeneration. D2R-/- mice develop seizures when administered KA doses that are not epileptogenic for wild-type (WT) littermates. The spatiotemporal pattern of c-fos and c-jun mRNA induction well correlates with the occurrence of seizures in D2R-/- mice. Moreover, KA-induced seizures result in extensive hippocampal cell death in D2R-/- but not WT mice. In KA-treated D2R-/- mice, hippocampal neurons die by apoptosis, as indicated by the presence of fragmented DNA and the induction of the proapoptotic protein BAX. These results reveal a central role of D2Rs in the inhibitory control of glutamate neurotransmission and excitotoxicity.

Fig. 1.

Expression of D2 and glutamate receptors in WT and D2R−/− mice. a, D2R binding sites in the hippocampus of WT and D2R−/− mice. b, High-affinity KA binding sites in the cerebral cortex and hippocampus of WT and D2R−/− mice.ctx, Cerebral cortex; CA3, CA3 hippocampal subfield; DG, dentate gyrus;slm, stratum lacunosum moleculare. Scale bar, 1.3 mm.c, Immunoblotting of glutamate receptor subunits (as indicated) on protein extracts from WT (+/+) and D2R−/− (−/−) hippocampi.

Fig. 2.

Severity of KA-induced seizures in WT, D2R+/−, D2R−/−, C57BL/6, and 129/Sv mice. Dose–response effect of KA treatments in the different genotypes tested, as indicated (n = 10 mice per genotype). Columns represent the maximum seizure rating scale value scored by each genotype over a period of 2 hr after KA administration. Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; Mann–Whitney U test between D2R−/− and each of the other genotypes.

Fig. 3.

Spatiotemporal pattern of IEGs induction in the brain of WT and D2R−/− mice treated with 20 mg/kg KA. c-fos (a) and c-jun(b) mRNA in situ hybridizations. Genotypes, treatments, and relevant brain areas are as indicated.CA1, CA3, Pyramidal cell layers of the hippocampus; ctx, cerebral cortex; DG, dentate gyrus; thal, thalamus. Scale bars, 1.8 mm.

Fig. 4.

KA-evoked seizures induce CA3 neuron apoptosis in D2R−/− but not WT mice. a, TUNEL staining of the whole dorsal hippocampus from mice killed 16 hr after KA treatment.b, High-power magnifications taken from the same sections shown in a. Arrowheads ina and b indicate the extension of TUNEL in a restricted part of the CA3 region. c, BAX immunoreactivity in CA3 pyramidal cells of WT and D2R−/− mice killed 16 hr after KA treatment. The absence of BAX labeling in the CA3 region of D2R−/− mice treated with 35 mg/kg KA (between arrowheads) corresponds to the region containing TUNEL-positive cells. Genotypes and treatments are as indicated. Scale bars:a, 300 μm; b, c, 70 μm.

Fig. 5.

Quantification of TUNEL-positive hippocampal CA3 cells in WT and D2R−/− mice treated with KA. Genotypes and treatments are as indicated. Values are expressed as the mean ± SEM of TUNEL-positive CA3 cells per area, as described in Materials and Methods. *p < 0.05, D2R−/− 20 mg/kg KA (KA20) treatment versus WT KA20 and D2R−/− saline; ***p < 0.0001, D2R−/− 35 mg/kg KA (KA35) treatment versus WT KA35 and D2R−/−KA20; Mann–Whitney U test;n = 5 animals per treatment group.

Fig. 6.

KA-evoked seizures induce CA3 cell damage in D2R−/− but not WT mice. a, Nissl staining of the whole dorsal hippocampus from mice killed 5 d after KA treatment.Asterisks indicate cell loss in a restricted part of the CA3 subfield in D2R−/− mice. b, High-power magnifications taken from the same sections shown in a.c, GFAP immunostaining of the CA3 region. Genotypes and treatments are as indicated. Scale bars: a, 270 μm;b, c, 70 μm.

Fig. 7.

IEGs induction in the brain of WT and D2R−/− mice treated with 35 mg/kg KA. c-fos(a) and c-jun(b) mRNA in situ hybridizations. Genotypes are as indicated. Scale bar, 1.2 mm.

Fig. 8.

KA-evoked seizures induce CA3 cell damage in D2R−/− but not WT congenic C56BL/6 mice. Nissl staining of the whole dorsal hippocampus from WT (a) and D2R−/− (b) mice killed 5 d after 35 mg/kg KA treatment. Asterisks indicate cell loss in a restricted part of the CA3 subfield in D2R−/− mice. Scale bar, 150 μm.