Alteration of rat hippocampal neurogenesis and neuronal nitric oxide synthase expression upon prenatal exposure to tamoxifen

Quantitative analysis of the effects of developmental stages and tamoxifen treatment on cell number. The number of cells was counted in relation to the subregion and age. (A) Tamoxifen significantly reduced the cell number in CA1 subregion to first week and demonstrated no effects in third week. (B) Tamoxifen did not alter the cell number in CA3 subregion in all ages. (C) Tamoxifen did not alter the cell number in D.G. subregion at either age. (D) Tamoxifen reduced the number of cells in subiculum to first week and demonstrated no effects on in third weeks. Data are expressed as mean±SEM. *P<0.05; **P<0.01 significantly different from Control.

Photomicrograph of Nissl staining of CA1 neurons of E22 (A1, A2), P1 (B1, B2), P7 (C1, C2), and P21 (D1, D2) of control (Control) and tamoxifen-treated (Experiment) animals. In the control groups the thickness of the layers increases from E22 to P21 and neurons are being matured. In the experimental groups damaged neurons (arrowheads) are heavily stained and dense nuclei with irregular nuclear membrane (short thin arrows), nuclear shrinkage (thick arrows), and chromatin clumping (long thin arrows) are marked. P21 is the least affected area and exhibited shrunken and triangulated neuronal body with a possible lightly reaction in the immediate vicinity. Majority of neurons in P21 area are morphologically unaltered and similar in form and staining to those in the control tissue. Photomicrographs are representative of n=8. Scale bars=250μm.

NADPH-diaphorase staining of CA1 subregion in prenatal E22. Thick arrow shows a neuron. Long thin arrow shows strong NADPH-d reaction. Short thin arrow shows weak NADPH-d reaction. Panel A: staining is shown for NADPH-d reaction in normal group, without treatment, in early development stage. Few neuron show high intense NADPH reaction (long thin arrow). Panel B: blank NADPH-d reaction in treatment group (thick arrow). Panel C: weak NADPH-d reaction in treatment group. (short thin arrow). Photomicrographs are representative of n=8. Scale bar=100μm.

NADPH-diaphorase staining of CA1 subregion in postnatal P1. Thick arrow shows a neuron. Long thin arrow shows strong NADPH-d reaction. Short thin arrow shows weak NADPH-d reaction. Panel A: NADPH-d reaction is shown in normal group, without treatment, in development stage. We demonstrated intense (weaker than E22) NADPH reaction (long thin arrow). Panel B: blank NADPH-d reaction in treatment group. There is no NADPH-d reaction (thick arrow). Panel C: NADPH-d reaction in treatment group is weak (short thin arrow). Photomicrographs are representative of n=8. Scale bar=100μm.

NADPH-diaphorase staining of CA1 in prenatal P7. Thick arrow shows a neuron. Long thin arrow shows strong NADPH-d reaction. Panel A: reactions are shown for NADPH-d in normal group, without treatment, in more development stage with more neurons. We demonstrated intense but weaker than P1 NADPH reaction (long thin arrow). Panel B: blank NADPH-d in treatment group with no NADPH-d reaction. A neuron is shown by thick arrow. Panel C: NADPH-d reaction in treatment group. We have irregular mild reaction (short thin arrow). Photomicrographs are representative of n=8. Scale bar=100μm.

NADPH-diaphorase staining of CA1 in prenatal P21. Thick arrow shows a neuron. Long thin arrow shows strong NADPH-d reaction. Short thin arrow shows weak NADPH-d reaction. Panel A: shows NADPH-d reaction in normal group, without treatment, in late development stage with much more neurons. We demonstrated low NADPH-d reaction (long thin arrow). Panel B: blank NADPH-d reaction in treatment group. A neuron is marked with think arrow with no NADPH-d reaction (thick arrow). Panel C: NADPH-d reaction in treatment group. We have intense reaction (short thin arrow). Photomicrographs are representative of n=8. Scale bar=100μm.

Immunofluoresent microscopy of nNOS expression in developing hippocampus. Expression of nNOS is shown for prenatal period of E22 (Panel A), postnatal P1 (Panel B), postnatal period P7 (Panel C), and postnatal period P21 (Panel D). Very low nNOS reaction in prenatal E22 is slightly increased in postnatal P1, further increased in postnatal P7, and highly increased 21 days after birth (P21).

Western blot analysis of hippocampal nNOS during developing hippocampus. Expression of nNOS in hippocampal CA1 region is shown for prenatal period of E22 (E22), postnatal P1 (P1), postnatal period P7 (P7), and postnatal period P21 (P21). Low nNOS expression at E22 increased during development and peaks at P21.