Identification of Dopaminergic Neurons of Nigral and Ventral Tegmental Area Subtypes in Grafts of Fetal Ventral Mesencephalon Based on Cell Morphology, Protein Expression, and Efferent Projections

Differential distribution of dopaminergic neuron subtypes in VM grafts and substantia nigra based on morphology. A-F, Six weeks after transplantation to the adult (A) or neonatal (D) striatum, GFP-immunopositive cells could be found in both central and peripheral locations within the graft core. Cells within the center of the graft (B, E) were rounded and significantly smaller, on average, than the more elongated and angular cells in the periphery (C, F). G-J, In the adult mouse mesencephalon (G), the TH⁺ neurons in the VTA (I), including those along the midline (H), are characteristically smaller and more rounded in shape compared with the larger and more angular TH⁺ cells within the SNpc (J). The scatter plots in K give the diameters of GFP⁺ cells in different regions of VM grafts placed in 6-OHDA-lesioned adults (filled circles) and TH⁺ cells in the intact mouse VTA and SNpc (open circles). The more medial/central cells were significantly smaller than those positioned more laterally/peripherally. The average cell diameters (indicated at the top of each group) differed significantly: Student's t test, ^*p < 0.01; n = 92 (VTA), n = 93 (SNpc), and n = 124 (center and periphery). Scale bars: A, D, 500 μm; B, C, E, F, H-J, 50 μm; G, 1 mm.

Calbindin and Girk2 expression in dopaminergic neurons in the adult mouse mesencephalon. Confocal images show the distribution of calbindin (A-C) and Girk2 (D-E) expressing cells within dopaminergic neurons of the substantia nigra from an adult mouse. Note that the calbindin (B) and Girk2 (C) expression domains appear distinctly nonoverlapping: calbindin is expressed predominately in the VTA and also in SN pars lateralis (C), whereas Girk2 expression is primarily restricted to the SNpc (F). i-v, High-magnification images showing the expression of calbindin and Girk2 within TH⁺ neurons of the VTA (i, ii), SNpc (iii, iv), and SN pars lateralis (v). The positions from which these images were taken are indicated by boxed areas in C and F. The dashed-boxed areas in A and B denote a small group of calbindin⁺/TH⁺ cells consistently seen in a dorsal region of the SNpc. Calbindin and Girk2 expression are not restricted to dopaminergic neurons and are found in many TH^- cells throughout the brain: note, for example, the prominent Girk2 expression in the red nucleus (RN). Scale bars: A-F, 500 μm; i-v, 50 μm.

Calbindin and Girk2 expression in dopaminergic cells of VM grafts. Confocal immunohistochemistry for GFP (A), Girk2 (B), and calbindin (C). Six weeks after transplantation into intact or 6-OHDA-lesioned adult rats, all VM grafts contained GFP⁺ neurons expressing Girk2 or calbindin (D). The example shown here is from an intact adult recipient. Girk2/GFP coexpressing cells were positioned predominately in the periphery of the graft, whereas the calbindin/GFP coexpressing cells were located mainly in the center. The boxed areas in A-D incorporate a peripheral and more central aspect of the graft and are shown at higher magnification (F-I). As is the case in the adult SN, the grafts contained also many nondopaminergic (GFP^-) calbindin- and Girk2-expressing cells (E, from dashed box in D). Quantitative analysis (J) showed that the peripheral regions of the graft contained significantly more Girk2⁺/GFP⁺ cells and the central regions significantly more calbindin⁺/GFP⁺ cells. Scale bars: A-D, 200 μm; F-I, 50 μm. Student's t test, ^*p < 0.01; n = 7 for all groups.

Graft-derived dopaminergic innervation of the forebrain in intact and denervated adult recipients. Six weeks after transplantation of E12.5 VM cells into the striatum of intact or 6-OHDA-lesioned adult rats, the graft-derived dopaminergic innervation of the host was assessed by GFP immunohistochemistry. The striatal volume innervated by the graft appeared consistently greater in lesioned (B) relative to intact (A) hosts, and formal quantification showed that this difference was significant (C, light gray bars). The volume of the graft core, as defined as the tissue occupied by GFP⁺ cell bodies, was not significantly different in intact and lesioned hosts (C, black bars). D gives an overview of the distribution of graft-derived GFP⁺ fibers, illustrated schematically in coronal sections (a-e) through one of the lesioned hosts, as indicated in the sagittal drawing. E-I give photographic examples of graft-derived GFP⁺ fibers in the striatal fiber bundles (E), frontal cortex (F, G), ventrolateral striatum (H), and the amygdala (I). The locations from which these images were taken are indicated in D. v, Ventricle. Scale bars: A, B, 1 mm; E, 100 μm; F-I, 100 μm. Student's t test, ^*p < 0.01).

Graft-derived dopaminergic innervation of the forebrain after transplantation in intact and lesioned neonatal recipients. GFP immunohistochemistry 6 weeks after transplantation. In the intact striatum, certain areas were innervated more extensively than others, giving a prominent patchy appearance throughout the structure (A). Innervation of the 6-OHDA-lesioned recipients was more extensive, resulting in a more homogenous fiber pattern throughout dorsal striatum (B). C gives an overview of GFP⁺ fiber innervation throughout the brain, illustrated schematically in coronal sections (a-e) through one of the lesioned hosts, as indicated in the sagittal drawing. D-I, Photographic examples are also given of GFP⁺ fibers in frontal cortex (D), piriform cortex (E), forceps minor (F), olfactory bulb (G), contralateral striatum and corpus callosum (H, boxed area shown at larger magnification as inset; arrowheads indicate GFP⁺ fibers in corpus callosum), and contralateral septum (I). The locations from which these images are taken are indicated in C. The image of GFP fibers in piriform cortex is taken from an animal in which the graft was placed quite ventrally, not represented in the schematic drawing. I, Cortex layer 1; III, cortex layer 3; v, ventricle. Scale bars: A, B, 1 mm; D, E, I, 200 μm; F, G, 500 μm; H, inset, 100 μm.

Retrograde tracing from dorsolateral striatum in VM grafted neonates. Six weeks after transplantation of wild-type VM tissue in 6-OHDA-lesioned neonates, CTB prelabeled with an Alexa-555 fluorophore was injected into the dorsolateral striatum. Immunohistochemistry for TH (A) and Girk2 (C) shows that many retrogradely labeled CTB⁺ cells (B) in the VM grafts expressed both of these proteins (D). Arrowheads in A-D identify the dopaminergic (TH⁺) cells that had incorporated the CTB tracer, located in the periphery of the graft. The boxed area is shown at higher magnification (E-H). In addition to TH⁺ cells (arrowhead), the CTB tracer was taken up by a number of TH^- cells (arrow) lying within the VM grafts (E-H). CTB staining resulting from passive diffusion of the tracer is illustrated at the injection site in the dorsolateral striatum (I) and at the level of the graft (J). g, Graft; v, ventricle. Scale bars: A-D, 200 μm; E-H, 50 μm; I, J, 500 μm.