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The A9 dopamine neuron component in grafts of ventral mesencephalon is an important determinant for recovery of motor function in a rat model of Parkinson's disease.

Grealish S, Jönsson ME, Li M, Kirik D, Björklund A, Thompson LH - Brain (2010)

Bottom Line: Here, we report results from a series of grafting experiments where the anatomical and functional properties of grafts either selectively lacking in A9 neurons, or with a typical A9/A10 composition were compared.The findings highlight dopamine neuronal subtype composition as a potentially important parameter to monitor in order to understand the variable nature of functional outcome better in transplantation studies.Furthermore, the results have interesting implications for current efforts in this field to generate well-characterized and standardized preparations of transplantable dopamine neuronal progenitors from stem cells.

View Article: PubMed Central - PubMed

Affiliation: Wallenberg Neuroscience Centre, Lund University, Lund, Sweden.

ABSTRACT
Grafts of foetal ventral mesencephalon, used in cell replacement therapy for Parkinson's disease, are known to contain a mix of dopamine neuronal subtypes including the A9 neurons of the substantia nigra and the A10 neurons of the ventral tegmental area. However, the relative importance of these subtypes for functional repair of the brain affected by Parkinson's disease has not been studied thoroughly. Here, we report results from a series of grafting experiments where the anatomical and functional properties of grafts either selectively lacking in A9 neurons, or with a typical A9/A10 composition were compared. The results show that the A9 component of intrastriatal grafts is of critical importance for recovery in tests on motor performance, in a rodent model of Parkinson's disease. Analysis at the histological level indicates that this is likely to be due to the unique ability of A9 neurons to innervate and functionally activate their target structure, the dorsolateral region of the host striatum. The findings highlight dopamine neuronal subtype composition as a potentially important parameter to monitor in order to understand the variable nature of functional outcome better in transplantation studies. Furthermore, the results have interesting implications for current efforts in this field to generate well-characterized and standardized preparations of transplantable dopamine neuronal progenitors from stem cells.

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The Pitx3 knockout phenotype. Immunohistochemistry for TH in the adult Pitx3WT/GFP (A, B) and Pitx3GFP/GFP (C, D) brain. In Pitx3GFP/GFP mice there was a distinct loss of TH+ neurons throughout the substantia nigra (compare A and C) and a corresponding loss of TH+ terminals in the dorsolateral striatum (compare B and D). Immunohistochemistry for TH (red), GFP (green) and PITX3 (blue) shows PITX3 expression in midbrain dopamine neurons in Pitx3WT/GFP mice (E) and lack of expression in the Pitx3GFP/GFP (Pitx3 ) mice (F). CPu = caudate-putamen unit; NAc = nucleus accumbens; SN = substantia nigra; VTA = ventral tegmental area. Scale: A and C, 200 µm; B and D, 500 µm; E–F, 50 µm.
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Figure 2: The Pitx3 knockout phenotype. Immunohistochemistry for TH in the adult Pitx3WT/GFP (A, B) and Pitx3GFP/GFP (C, D) brain. In Pitx3GFP/GFP mice there was a distinct loss of TH+ neurons throughout the substantia nigra (compare A and C) and a corresponding loss of TH+ terminals in the dorsolateral striatum (compare B and D). Immunohistochemistry for TH (red), GFP (green) and PITX3 (blue) shows PITX3 expression in midbrain dopamine neurons in Pitx3WT/GFP mice (E) and lack of expression in the Pitx3GFP/GFP (Pitx3 ) mice (F). CPu = caudate-putamen unit; NAc = nucleus accumbens; SN = substantia nigra; VTA = ventral tegmental area. Scale: A and C, 200 µm; B and D, 500 µm; E–F, 50 µm.

Mentions: In order to generate mice with GFP knocked into either one (Pitx3WT/GFP) or both (Pitx3GFP/GFP) Pitx3 alleles, Pitx3GFP/GFP males were bred with Pitx3WT/GFP females. This yielded mixed litters at the expected Mendelian frequency of approximately 50% for each genotype. In agreement with the original report by Maxwell and colleagues (2005), immunohistological analysis of the adult midbrain confirmed that PITX3 expression was present in Pitx3WT/GFP mice but completely abolished in Pitx3GFP/GFP littermates (Fig. 2E and F). While heterozygous Pitx3WT/GFP mice appeared indistinguishable from wild-type animals at the behavioural and histological level (not shown), the Pitx3GFP/GFP knockouts displayed a phenotype which is very similar, if not identical, to the previously described aphakia mice (Nunes et al., 2003; Smidt et al., 2004). The knockout animals were smaller in size than Pitx3WT/GFP littermates, had a small eye phenotype and histological analysis revealed a prominent loss of dopamine neurons within the substantia nigra pars compacta (Fig. 2A and C). This pattern of cell loss was reflected at the level of the striatum, where there was an almost complete loss of innervation of areas normally innervated by the A9 midbrain dopamine neurons, most notably the dorsolateral striatum, while areas innervated by A10 midbrain dopamine neurons, such as nucleus accumbens and adjacent areas of the ventromedial striatum, appeared relatively intact (Fig. 2B and D). The selective loss of A9 neurons in the Pitx3GFP/GFP knockouts was also apparent based on expression of GIRK2 and Calbindin, which are largely confined to the A9 and A10 populations, respectively (Fig. 3). While there was an almost complete loss of the GFP+/GIRK2+ midbrain dopamine population throughout the substantia nigra pars compacta in the Pitx3GFP/GFP mice (Fig. 3C and D), the GFP+/Calbindin+ midbrain dopamine neurons in the ventral tegmental area were largely spared (Fig. 3E and F). The GIRK2+ neurons present in the dorsolateral part of the ventral tegmental area were also spared (Fig. 3D).


The A9 dopamine neuron component in grafts of ventral mesencephalon is an important determinant for recovery of motor function in a rat model of Parkinson's disease.

Grealish S, Jönsson ME, Li M, Kirik D, Björklund A, Thompson LH - Brain (2010)

The Pitx3 knockout phenotype. Immunohistochemistry for TH in the adult Pitx3WT/GFP (A, B) and Pitx3GFP/GFP (C, D) brain. In Pitx3GFP/GFP mice there was a distinct loss of TH+ neurons throughout the substantia nigra (compare A and C) and a corresponding loss of TH+ terminals in the dorsolateral striatum (compare B and D). Immunohistochemistry for TH (red), GFP (green) and PITX3 (blue) shows PITX3 expression in midbrain dopamine neurons in Pitx3WT/GFP mice (E) and lack of expression in the Pitx3GFP/GFP (Pitx3 ) mice (F). CPu = caudate-putamen unit; NAc = nucleus accumbens; SN = substantia nigra; VTA = ventral tegmental area. Scale: A and C, 200 µm; B and D, 500 µm; E–F, 50 µm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2822634&req=5

Figure 2: The Pitx3 knockout phenotype. Immunohistochemistry for TH in the adult Pitx3WT/GFP (A, B) and Pitx3GFP/GFP (C, D) brain. In Pitx3GFP/GFP mice there was a distinct loss of TH+ neurons throughout the substantia nigra (compare A and C) and a corresponding loss of TH+ terminals in the dorsolateral striatum (compare B and D). Immunohistochemistry for TH (red), GFP (green) and PITX3 (blue) shows PITX3 expression in midbrain dopamine neurons in Pitx3WT/GFP mice (E) and lack of expression in the Pitx3GFP/GFP (Pitx3 ) mice (F). CPu = caudate-putamen unit; NAc = nucleus accumbens; SN = substantia nigra; VTA = ventral tegmental area. Scale: A and C, 200 µm; B and D, 500 µm; E–F, 50 µm.
Mentions: In order to generate mice with GFP knocked into either one (Pitx3WT/GFP) or both (Pitx3GFP/GFP) Pitx3 alleles, Pitx3GFP/GFP males were bred with Pitx3WT/GFP females. This yielded mixed litters at the expected Mendelian frequency of approximately 50% for each genotype. In agreement with the original report by Maxwell and colleagues (2005), immunohistological analysis of the adult midbrain confirmed that PITX3 expression was present in Pitx3WT/GFP mice but completely abolished in Pitx3GFP/GFP littermates (Fig. 2E and F). While heterozygous Pitx3WT/GFP mice appeared indistinguishable from wild-type animals at the behavioural and histological level (not shown), the Pitx3GFP/GFP knockouts displayed a phenotype which is very similar, if not identical, to the previously described aphakia mice (Nunes et al., 2003; Smidt et al., 2004). The knockout animals were smaller in size than Pitx3WT/GFP littermates, had a small eye phenotype and histological analysis revealed a prominent loss of dopamine neurons within the substantia nigra pars compacta (Fig. 2A and C). This pattern of cell loss was reflected at the level of the striatum, where there was an almost complete loss of innervation of areas normally innervated by the A9 midbrain dopamine neurons, most notably the dorsolateral striatum, while areas innervated by A10 midbrain dopamine neurons, such as nucleus accumbens and adjacent areas of the ventromedial striatum, appeared relatively intact (Fig. 2B and D). The selective loss of A9 neurons in the Pitx3GFP/GFP knockouts was also apparent based on expression of GIRK2 and Calbindin, which are largely confined to the A9 and A10 populations, respectively (Fig. 3). While there was an almost complete loss of the GFP+/GIRK2+ midbrain dopamine population throughout the substantia nigra pars compacta in the Pitx3GFP/GFP mice (Fig. 3C and D), the GFP+/Calbindin+ midbrain dopamine neurons in the ventral tegmental area were largely spared (Fig. 3E and F). The GIRK2+ neurons present in the dorsolateral part of the ventral tegmental area were also spared (Fig. 3D).

Bottom Line: Here, we report results from a series of grafting experiments where the anatomical and functional properties of grafts either selectively lacking in A9 neurons, or with a typical A9/A10 composition were compared.The findings highlight dopamine neuronal subtype composition as a potentially important parameter to monitor in order to understand the variable nature of functional outcome better in transplantation studies.Furthermore, the results have interesting implications for current efforts in this field to generate well-characterized and standardized preparations of transplantable dopamine neuronal progenitors from stem cells.

View Article: PubMed Central - PubMed

Affiliation: Wallenberg Neuroscience Centre, Lund University, Lund, Sweden.

ABSTRACT
Grafts of foetal ventral mesencephalon, used in cell replacement therapy for Parkinson's disease, are known to contain a mix of dopamine neuronal subtypes including the A9 neurons of the substantia nigra and the A10 neurons of the ventral tegmental area. However, the relative importance of these subtypes for functional repair of the brain affected by Parkinson's disease has not been studied thoroughly. Here, we report results from a series of grafting experiments where the anatomical and functional properties of grafts either selectively lacking in A9 neurons, or with a typical A9/A10 composition were compared. The results show that the A9 component of intrastriatal grafts is of critical importance for recovery in tests on motor performance, in a rodent model of Parkinson's disease. Analysis at the histological level indicates that this is likely to be due to the unique ability of A9 neurons to innervate and functionally activate their target structure, the dorsolateral region of the host striatum. The findings highlight dopamine neuronal subtype composition as a potentially important parameter to monitor in order to understand the variable nature of functional outcome better in transplantation studies. Furthermore, the results have interesting implications for current efforts in this field to generate well-characterized and standardized preparations of transplantable dopamine neuronal progenitors from stem cells.

Show MeSH
Related in: MedlinePlus