Figure 1.
hTH-GFP expression correlates with TH expression in the adult midbrains of Line 12141.
TH staining (A) co-labels with GFP (B) expression in midbrain areas, especially in the cell bodies (C) of the SNpc and VTA. The inset of Panel (C) shows that GFP antibody can detect GFP signals in the fine dendritic processes emanating from the cell bodies into the SN pars reticulata (SNpr), which are not as easily visualized by GFP fluorescence in Panel C.
Figure 2.
hTH-GFP expression correlates with TH expression in some but not all DA target regions.
TH staining co-labels with GFP expression in adult striatum (A-A″) and olfactory bulb (B-B″) in Line 12141. However, GFP expression was less evident in the hypothalamus (C-C″) and absent in the locus coeruleus (D) despite robust TH staining. Ectopic GFP expression is seldom observed in non-TH expressing areas such as habenular nucleus (E) and supraoptic nucleus (F) in Line 12141.
Table 1.
Comparison of TH and GFP expression throughout the CNS and PNS in various hTH-GFP transgenic rat lines.
Figure 3.
Expression of mDA neuronal markers in hTH-GFP rats at various developmental stages.
The mDA progenitor marker Foxa2 (A) is widely distributed along the floor plate of the midbrain at E11.5 while GFP (A′) is detected in only a few midline cells in this region. By E12.5, Foxa2 (B), Lmx1a (C) and TH (D) are all expressed in the central portion of the ventral midbrain. However, Foxa2 expression extends more laterally than Lmx1a. TH expression co-labels with GFP but double-labeled cells are seen only at the base of the midline zone. At all stages examined: E14.5 (E), E18.5 (F) and P3 (G), GFP expression closely approximates TH expression in the midbrain. Scale bar = 50 µm in A, C, D. Scale bar = 100 µm in B, E, F, G.
Figure 4.
FACS sorting of midbrain DA neurons from E14.5 hTH-GFP rats of Line 12141.
(A) Raw data of sorted cells. Gating for live GFP+ cells was established by utilizing positive controls for GFP and propidium iodide (PI) stained cells (not shown). The gate, P2, excluded weakly GFP+ cells and any PI+ (dead) cells. This allowed for the collection of approximately 90% of GFP+ cells (B). Collected cells were plated at a density of 300,000cells/well and grown for 3 days, fixed and immunostained for TH (C) exhibit similar co-labeling seen in vivo (Figure 1).
Figure 5.
Treatment with MPP+ demonstrates selective hTH-GFP+ neuron death in the SN in vitro.
E14.5 hTH-GFP+ SN and VTA neurons were plated at a density of 300,000cells/well and grown for 7 days in defined media. (A) Control SN neurons were then treated with media+PBS. (B-C) SN neurons were treated with MPP+ at 10 and 50 µM for 48 hours. TH+ neurons treated with 10 µM MPP+ (B) show healthy, undamaged morphology, while 50 µM (C) treated SN neurons are significantly degenerated compared to control (p<0.05). Control (not shown) or 50 µM MPP+ treated (D) VTA neurons exhibit normal, healthy morphology without a significant decline in TH+ cell number.
Figure 6.
6-OHDA-treated hTH-GFP reporter rat model of PD in vivo.
Six weeks after 6-OHDA lesion, GFP or TH expression is undetected on the lesion side while both GFP and TH expression is unaffected in the striatum (A) and midbrain (B) on the contralateral side. Rotation tests following 5 mg/kg ip amphetamine challenge were performed at 3 weeks and 6 weeks after lesion. The scores are listed and plotted in Panel (C). In 9 of 10 cases, rats exhibit a robust lesion with over 50 full clockwise turns per 5 minutes. Many rats reached maximum level after 3 weeks (rats 3,4,6,7,9,10), others after 6 weeks (rats 1,2,8). Only one rat (rat 5) showed spontaneous recovery of function after 6 weeks.
Figure 7.
Transplantation of E14.5 hTH-GFP midbrain cells into the striatum of wild type 6-OHDA-lesioned rats.
(A–B) At 4 days after transplantation, grafted cells are found along the needle track and at the three cell deposit sites. The majority of transplanted cells are TH+ (A) and Foxa2+ (B). In addition to GFP+ cells, Foxa2+/GFP- dopaminergic progenitor cells are also found in the graft (B). (C) Importantly, TH+/GFP+ processes are seen in the striatum on the ipsilateral lesioned side 4 weeks after transplantation (unlike non-transplanted controls- see Fig. 6A). The magnified view of white box is shown in (D′) and (D″). This section is rostral to the needle track, thus the extensive network of TH+ processes despite the presence of only a few grafted cells. (E) Rotation test scores following 5mg/kg ip amphetamine challenge substantially decreased in all 4 rats 3 weeks after transplantation, consistent with functional recovery from transplantation of fetal hTH-GFP+ mDA neurons; * p<0.05, paired samples t-test.
Table 2.
Rotation scores and Detected TH+ cells in grafts of 4 transplanted rats.