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. 2000 Dec 25;151(7):1537–1548. doi: 10.1083/jcb.151.7.1537

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© 2000 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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Characterization of transgenic midbrain dopaminergic neurons and protection against neurotoxin-induced degeneration. (A) Expression of the Ras-TG in dopaminergic neurons of the substantia nigra. (I) A coronal section through the midbrain at the level of the substantia nigra was hybridized with a lacZ riboprobe and showed staining in a subset of neurons. (II) Immunohistochemical analysis of the same section using a monoclonal antibody against TH (Roche) and a Texas red–labeled secondary antibody shows TH expression in the neuron expressing the transgene. Bar, 20 μm. (III) Overlay of the immunofluorescence picture and the brightfield micrograph, indicating that the Ras-TG transcript is found in the TH-positive neuron. (B) Effect of 6-OHDA or MPP⁺ treatment on the number of cultured midbrain dopaminergic neurons derived from synRas-TG mice or their wt siblings. Midbrain dopaminergic neurons were derived from E14 embryos and cultured under serum-free conditions. After treatment with MPP⁺ (1 μM) for 36 h or 6-OHDA (150 μM) for 90 min, cells were fixed with methanol. Dopaminergic neurons were identified using a monoclonal antibody against TH (Roche), a biotin-labeled secondary antibody (Sigma-Aldrich), and a streptavidine–FITC conjugate. After counting dopaminergic neurons, the genotype was determined by Southern blot analysis. Treatment with the toxin resulted in degeneration of the dopaminergic neurons. This toxin-induced degeneration was markedly reduced in cultures derived from synRas-TG mice. The numbers of nondopaminergic neurons remained unchanged (data not shown). (C) Determination of dopamine after in vivo treatments with MPTP in synRas-TG mice and their wt siblings (see Materials and Methods). In the synRas-TG mice, the MPTP-induced decrease of dopamine contents was profoundly reduced.

Characterization of transgenic midbrain dopaminergic neurons and protection against neurotoxin-induced degeneration. (A) Expression of the Ras-TG in dopaminergic neurons of the substantia nigra. (I) A coronal section through the midbrain at the level of the substantia nigra was hybridized with a lacZ riboprobe and showed staining in a subset of neurons. (II) Immunohistochemical analysis of the same section using a monoclonal antibody against TH (Roche) and a Texas red–labeled secondary antibody shows TH expression in the neuron expressing the transgene. Bar, 20 μm. (III) Overlay of the immunofluorescence picture and the brightfield micrograph, indicating that the Ras-TG transcript is found in the TH-positive neuron. (B) Effect of 6-OHDA or MPP⁺ treatment on the number of cultured midbrain dopaminergic neurons derived from synRas-TG mice or their wt siblings. Midbrain dopaminergic neurons were derived from E14 embryos and cultured under serum-free conditions. After treatment with MPP⁺ (1 μM) for 36 h or 6-OHDA (150 μM) for 90 min, cells were fixed with methanol. Dopaminergic neurons were identified using a monoclonal antibody against TH (Roche), a biotin-labeled secondary antibody (Sigma-Aldrich), and a streptavidine–FITC conjugate. After counting dopaminergic neurons, the genotype was determined by Southern blot analysis. Treatment with the toxin resulted in degeneration of the dopaminergic neurons. This toxin-induced degeneration was markedly reduced in cultures derived from synRas-TG mice. The numbers of nondopaminergic neurons remained unchanged (data not shown). (C) Determination of dopamine after in vivo treatments with MPTP in synRas-TG mice and their wt siblings (see Materials and Methods). In the synRas-TG mice, the MPTP-induced decrease of dopamine contents was profoundly reduced.