TH immunostaining of representative sections from mDA neurons of E18.5 mice.

<p>Normal differentiation of mDA into SNc and VTA (a, b) and normal distribution of RRF neurons (c) in wildtype. In A/V dko there was nigral neuron loss and accumulation of TH cells in VTA (d, e, arrow) in addition to the alteration in distribution and reduction in numbers of RRF neurons (f, arrow). Scale bars 100 µm in a, b, c, d and 200 µm in c, f.</p

Overview of mutant mice lines.

<p>Overview of mutant mice lines.</p

Reduction of nigral dopaminergic neurons in both ApoER2 and VLDLr mutant mice at P25.

<p>TH staining of mDA neurons of wildtype (a, e) and single ApoER2 (c) and VLDLr (g) mutant mice defined by dotted black line. (b, d, f, h) Higher magnification images for the areas defined by dotted black lines in (a, c, e, g) reveal a slight reduction of TH-positive neurons of SNc in ApoER2^−/− (d) and a pronounced reduction in VLDLr^−/− (h) as compared to corresponding wildtype littermate controls (b, f). Scale bars  = 200 µm. Stereological estimates of the total numbers of TH-positive neurons in SNc in ApoER2 animals (I), SNc, VTA and total numbers of mDA neurons (j, k and l respectively) in VLDLr animals, reveal about 15% and 40% reduction in ApoER2^−/− (i) and VLDLr^−/− (j) respectively, as compared to the wildtype animals, and significant increase of VTA neuron number in VLDLr^−/− (k). There was no significant reduction in the total numbers of mDA neurons between VLDLr^+/+ and VLDLr^−/− (l). Three animals were used for each genotype for cell counting. Results were given as means ± SEM. P-values derived from unpaired t test are ***p<0.0007 in i; ***p<0.0003 in j; ***p<0.0001 in k.</p

Loss of nigral dopaminergic neurons in A/V dko mice at P15.

<p>TH immunostaining of representative sections from mDA neurons of wildtype (a, b) and A/V dko (c, d). The rostral and intermediate sections of SNc in wildtype reveal correct positioning and normal migration of nigral neurons. However, in A/V dko the nigral neurons in the rostral sections appear irregularly organized (c), in addition to a pronounced loss of SNc neurons, which appear located lateral to VTA neurons (d). (e, f) Stereological counting of the total numbers of TH- positive neurons in SNc and VTA respectively reveals a significant reduction in the number of nigral neurons of A/V dko as compared to wildtype (e). However, in (f) a pronounced increase in VTA neuron numbers of A/V dko is seen. Three animals were used for each genotype for cell counting. Results are given as mean ± SEM. P-value derived from unpaired t-test are ***p<0.0001. Scale bar  = 200 µm.</p

Detection of active caspase-3 in the mesencephalic dopaminergic neurons.

<p>(a–j) Coronal paraffin brain sections of ApoER2/VLDLr^−/− (a–c), ApoER2^−/− (e–g) and VLDLr^−/− mice (h–J), stained with anti-active caspase-3 antibody (red) and TH (green). In all mutant mice (b, f, i) no positive signals for active caspase-3 were detected in SNc (a, e, h). (d) Positive control for active caspase-3: cells of intestinal villi, nuclei counterstained with Dapi (blue). (c, g, j) Merge of images in a, b; e, f, and h, i respectively. SN: Substania nigra, VTA: Ventral tegmental area. Scale bars = 200 µm.</p

Analysis of striatal TH fibre density at P15.

<p>TH staining of striatal sections of wildtype (a) and A/V dko (b). The TH staining of striatum indicates normal projections of SNc neurons despite their aberrant positioning in the VTA in A/V dko. Scale bar  = 500 µm. (c) Measuring the optical density of TH-positive fibres reveals no significant difference between wildtype and mutant mice.</p

Analysis of the caudal sections of mDA neurons.

<p>(a–f) Coronal brain sections stained with TH (green) and calbindin (red) of VLDLr^+/+ and VLDLR^−/− mice. (a–c) in VLDLr^+/+ mice, all TH positive neurons of VTA coexpress calbindin. In VLDLr^−/− (d–f) there were more TH positive cells in VTA than in VLDLr^+/+ VTA and not all TH neurons coexpress calbindin. Scale bar = 100 µm.</p

Analysis of the intermediate sections of mDA neurons in VLDLr^−/−.

<p>(a–f) Midbrain coronal sections stained with TH (green) and calbindin (red). (a, d) TH staining of VTA neurons (a) and the line of demarcation between VTA and SNc. (b, e) Calbindin immunostaining of mDA neurons of VLDLr^−/− mice. (c, f) Merge of images in a, b and d, e. Not all VTA neurons (c) and also nigral neurons (f) were positive for calbindin. Scale bar  = 100 µm.</p

Analysis of mDA neurons in Dab1−/− mice.

<p>TH immunostaining of representative sections of rostral sections of SN in wildtype (a) and Dab1 knockout littermates (b). (e, i) Higher magnification images reveal correct positioning, normal distribution and morphology of dopaminergic neurons in the rostral sections of wildtype mice. (f, j) show slight defect in the migration of the rostral sections of SNc neurons in Dab1 mutant mice. TH immunostaining of mDA neurons in intermediate sections in both wildtype (c) and Dab1^−/− mice (d). (g, h) Higher magnification images reveal significant reduction of nigral dopaminergic neurons in Dab1^−/− (h) as compare to wildtype (g). TH immunostaining of the caudal mDA neurons showing higher numbers of TH positive neurons in VTA of Dab1^−/− (k) as compare to Dab1^+/+ (m). Scale bars = 200 µm.</p

Analysis of RRF of mDA neurons at P15.

<p>TH staining of representative RRF coronal sections of wildtype (a, b), A/V dko (c) and Dab1^−/− (d) mice. (a, b) show the correct positioning and normal distribution of RRF in the wildtype. However, (c, d) reveal a severe alteration in the distribution of dopaminergic neurons of RRF and a reduction in numbers in A/V dko and Dab1−/−. Scale bar  = 500 µm.</p