Characterization of the Dopamine Defect in Primary Cultures of Dopaminergic Neurons from Hypoxanthine Phosphoribosyltransferase Knockout Mice  Doug W.

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Characterization of the Dopamine Defect in Primary Cultures of Dopaminergic Neurons from Hypoxanthine Phosphoribosyltransferase Knockout Mice  Doug W. Smith, Theodore Friedmann  Molecular Therapy  Volume 1, Issue 5, Pages 486-491 (May 2000) DOI: 10.1006/mthe.2000.0057 Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 1 Photomicrographs showing DIV 15, dopamine neurons (TH-immunostained) from wild- type (A and B) and HPRT-deficient mesencephalic cultures (C and D), under control (A and C) and GDNF- supplemented conditions (B and D). Note the absence of obvious morphological differences between the dopamine neurons of the two genotypes and the similar survival and neurite outgrowth- promoting effects of GDNF. Molecular Therapy 2000 1, 486-491DOI: (10.1006/mthe.2000.0057) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 2 Survival curves for dopamine neurons for wild-type and HPRT-deficient cultures under control and GDNF-supplemented conditions. Significant differences between control and GDNF-treated groups were apparent at days 5, 10, and 15 (in all cases P < 0.0001) but not at day 1. *** and ^^^ denote significant differences for wild-type and HPRT-deficient cultures, respectively. Thus, there was an interaction between treatment and DIV (F = 48.82, P < 0.0001) but not for genotype (F = 2.23, P < 0.1384) or any other interaction. Molecular Therapy 2000 1, 486-491DOI: (10.1006/mthe.2000.0057) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 3 Dopamine uptake response at different time points for control and GDNF-supplemented wild-type and HPRT-deficient cultures. Statistical analysis revealed significant effects of genotype (F = 31.46, P < 0.0001), treatment (F = 41.12, P < 0.0001), and DIV (F = 207.33, P < 0.0001) and an interaction between treatment and DIV (F = 3.34, P < 0.05) but not between any other factors. # denotes significant (P < 0.05) difference in uptake between wild-type and HPRT-deficient GDNF-treated dopamine neurons. Uptake in control cultures was not significantly different between genotypes; dopamine uptake in wild-type cultures was significantly higher at DIV 5, 10, and 15 (F = 5.98, P < 0.05; F = 5.35, P < 0.05; F = 5.98, P < 0.05), but not at DIV 1 (F = 2.23, P < 0.16). Molecular Therapy 2000 1, 486-491DOI: (10.1006/mthe.2000.0057) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

FIG. 4 Dopamine content at DIV 5, 10, and 15 for control and GDNF-sup-plemented wild-type and HPRT-deficient dopamine neurons. Dopamine levels at DIV 1 were too low for reliable detection. Statistical analysis revealed significant main effects for genotype (F = 21.13, P < 0.0001), treatment (F = 123.3, P < 0.0001), and DIV (F = 35.3, P < 0.0001) but no interaction between any of these. Comparison of dopamine contents between wild-type and HPRT-deficient neurons at the three time points for GDNF and control conditions revealed significantly higher dopamine levels in wild-type GDNF-treated dopaminergic neurons compared to HPRT-deficient counterparts at DIV 5 and 10. The difference at DIV 15 did not reach statistical significance. There were no differences in dopamine content between genotypes under control conditions. # denotes significant (P < 0.05) difference between GDNF-treated wild- type and HPRT-deficient dopaminergic neurons. Molecular Therapy 2000 1, 486-491DOI: (10.1006/mthe.2000.0057) Copyright © 2000 American Society for Gene Therapy Terms and Conditions

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