Transplantation site influences the phenotypic differentiation of dopamine neurons in ventral mesencephalic grafts in Parkinsonian rats

Abstract

Foetal midbrain progenitors have been shown to survive, give rise to different classes of dopamine neurons and integrate into the host brain alleviating Parkinsonian symptoms following transplantation in patients and animal models of the disease. Dopamine neuron subpopulations in the midbrain, namely A9 and A10, can be identified anatomically based on cell morphology and ascending axonal projections. G protein-gated inwardly rectifying potassium channel Girk2 and the calcium binding protein Calbindin are the two best available histochemical markers currently used to label (with some overlap) A9- and A10-like dopamine neuron subtypes, respectively, in tyrosine hydroxylase expressing neurons both in the midbrain and grafts. Both classes of dopamine neurons survive in grafts in the striatum and extend axonal projections to their normal dorsal and ventral striatal targets depending on phenotype. Nevertheless, grafts transplanted into the dorsal striatum, which is an A9 input nucleus, are enriched for dopamine neurons that express Girk2. It remains to be elucidated whether different transplantation sites favour the differential survival and/or development of concordant dopamine neuron subtypes within the grafts. Here we used rat foetal midbrain progenitors at two developmental stages corresponding to a peak in either A9 or A10 neurogenesis and examined their commitment to respective dopaminergic phenotypes by grafting cells into different forebrain regions that contain targets of either nigral A9 dopamine innervation (dorsal striatum), ventral tegmental area A10 dopamine innervation (nucleus accumbens and prefrontal cortex), or only sparse dopamine but rich noradrenaline innervation (hippocampus). We demonstrate that young (embryonic day, E12), but not older (E14), mesencephalic tissue and the transplant environment influence survival and functional integration of specific subtypes of dopamine neurons into the host brain. We also show that irrespective of donor age A9-like, Girk2-expressing neurons are more responsive to environmental cues in adopting a dopaminergic phenotype during differentiation post-grafting. These novel findings suggest that dopamine progenitors use targets of A9/A10 innervation in the transplantation site to complete maturation and the efficacy of foetal cell replacement therapy in patients may be improved by deriving midbrain tissue at earlier developmental stages than in current practice.

Keywords: A9 neuron; Cell transplantation; Dopamine; Girk2; Parkinson's disease; Striatum; Ventral mesencephalon.

Fig. 1

VM grafts affect drug-induced rotational bias in the dSTR (A) and N.Acc (B) groups. Mean (ipsilateral minus contralateral) rotation score over 90 min following an i.p. injection of 2.5 mg/kg metamphetamine in lesioned and grafted rats in the E12 and E14 groups. Data are collapsed across the secondary graft factor. (A) Both donor age graft groups produced a recovery of the lesion-induced behavioural deficit in the dSTR graft group. There was a significant change from ipsilateral rotation observed post-lesion to a net contralateral rotation post-graft, the classic over-compensatory response. (B) Animals in the E14 N.Acc graft group exhibited enhanced (*) ipsilateral rotational behaviour post-graft. Error bars indicate ± SEM, significance levels: * or †p < 0.05, *** or ###p < 0.001.

Fig. 2

Transplantation site does not influence total neuron yield in VM grafts in the Parkinsonian brain. (A–D) Photomicrographs illustrating NeuN staining of E12 VM grafts in different transplantation targets: (A) dSTR, (B) N.Acc, (C) PFC, and (D) HPC. There is a clear boundary between the graft and the surrounding host brain. Neurons are homogeneously distributed within the graft and their density appears to be higher in the graft than in the surrounding brain tissue. (A′) High magnification image from (A), illustrating the morphology of the NeuN-ir cells within the graft. (E) Quantification of NeuN-ir cells at 6 weeks post-graft revealed no significant differences between the donor age groups and transplantation site groups (n.s.). Columns depict group means; error bars illustrate ± SEM. Scale: A–D, 500 μm; A′: 50 μm. Abbreviations: dorsal striatum (dSTR), nucleus accumbens (N.Acc), prefrontal cortex (PFC), hippocampus (HPC).

Fig. 3

Immunohistochemical analysis of dopamine neuron survival in VM grafts in a rat model of PD. Photomicrographs of TH staining in E12 VM grafts in the dSTR (A), N.Acc (B), PFC (C), and HPC (D), and E14 VM grafts in the same cerebral targets (E, F, G, and H, respectively) at 6 weeks post-transplantation illustrating abundant dopamine neuron survival and integration into the host brain. (I) Photomicrograph of TH staining in a coronal section through 6-OHDA lesioned midbrain demonstrating extensive ablation of the host dopaminergic midbrain pathways. (J) High magnification image illustrating the morphology of TH-ir neurons within the transplant and graft-derived dopaminergic axons extended into the surrounding tissue. Grafts contained larger elongated TH-ir neurons suggestive of an A9-like phenotype (black arrow head) as well as smaller spherical neurons indicative of A10-like phenotype (grey arrow head). Scale bars: 500 μm (A–I) and 20 μm (J). Abbreviations: dorsal striatum (dSTR), nucleus accumbens (N.Acc), prefrontal cortex (PFC), hippocampus (HPC).

Fig. 4

Donor tissue age and transplantation site influence the number and density of TH-ir neurons in 6-week-old VM grafts in a rat model of PD. Data portray the total number of TH-ir neurons (A) and the percentage of TH-ir/NeuN-ir cells (B) in the grafts, graft volumes (C) and the density of TH-ir neurons in the grafts (D). Columns depict group means; error bars illustrate ± SEM; significance levels: *p < 0.05, ** or ‡‡p < 0.01, ***p < 0.001. Abbreviations: dorsal striatum (dSTR), nucleus accumbens (N.Acc), prefrontal cortex (PFC), hippocampus (HPC).

Fig. 5

Transplantation site influences A9-like dopamine neuron specification in VM grafts. Coronal sections through E12 VM grafts illustrating TH-ir (green) neurons co-expressing Girk2 (red: A–D) or Calbindin (red: E–H) in grafts in the dSTR (A, E), N.Acc (B, F), PFC (C, G) and HPC (D, H). Note the increase in the Girk2-ir/TH-ir neuron population in grafts in the dSTR compared to other grafts. (A′ and F′) High magnification images from (A and F), illustrating the co-localisation of Girk2 (A′) and Calbindin (F′) with TH and morphology of double labelled neurons within the transplant. (I) Total number of Girk2-ir/TH-ir neurons and (J) Calbindin-ir/TH-ir neurons within the grafts at each donor age and transplantation site. (K) Quantification of the proportion of Girk2-ir/TH-ir neurons and (L) Calbindin-ir/TH-ir neurons out of total TH-ir neurons within the grafts. The presence of targeted midbrain innervation of the transplantation site significantly increased the number and proportion of A9-like neurons in the grafts. Scale bars: 100 μm (A–H) and 25 μm (A′ and F′). Columns depict group means; error bars illustrate ± SEM; significance levels: *p < 0.05, ***p < 0.001. Abbreviations: dorsal striatum (dSTR), nucleus accumbens (N.Acc), prefrontal cortex (PFC), hippocampus (HPC).

Fig. 6

Transplantation site influences distribution of A9- and A10-like dopamine neurons within VM grafts. Percentages of Girk2-ir/TH-ir and Calbindin-ir/TH-ir neurons in the periphery and in the centre of E12 (A) and E14 grafts (B) at 6 weeks post-transplantation. Note the decrease in the percentage of Girk2-ir/TH-ir neurons in the periphery of grafts in HPC compared to grafts in other brain regions. The presence of A10 dopamine innervation of the N.Acc significantly increased the percentage of A10-like neurons in the periphery of the graft compared to the graft core in E14 group. Columns depict group means; error bars illustrate ± SEM; significance levels: *p < 0.05, **p < 0.01, ***p < 0.001 and accordingly for other symbols; different symbols are used to depict significant differences between distinct groups as follows: *centre vs periphery for Girk2/TH %, #centre vs periphery for Calbindin/TH % [special case (#) p < 0.1], †differences between Girk2/TH % and Calbindin/TH %, ‡differences between transplantation sites. Abbreviations: centre (C), periphery (P), dorsal striatum (dSTR), nucleus accumbens (N.Acc), prefrontal cortex (PFC), hippocampus (HPC).