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First published online 4 July 2007
doi: 10.1242/dev.002329

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Midbrain dopaminergic neurogenesis and behavioural recovery in a salamander lesion-induced regeneration model

Clare L. Parish^1,*, Anna Beljajeva^2,*, Ernest Arenas^1, and András Simon^2,,

¹ Department of Medical Biochemistry and Biophysics, Karolinska Institute, Center of Excellence in Developmental Biology, Stockholm, Sweden.
² Department of Cell and Molecular Biology, Medical Nobel Institute, Center of Excellence in Developmental Biology, Karolinska Institute, Stockholm, Sweden.

View larger version (78K): [in this window] [in a new window]   Fig. 1. Stereotaxic 6-OHDA injection leads to loss of DA cells in the diencephalon and the mesencephalon. (A,B) Low- and high-power photomicrographs of TH+ cells in the tegmental area of the newt midbrain. (C) TH+ fibres in the striatum from a sham-lesioned animal. (D) High-power photomicrograph of the boxed area in C. (E,F) TH+ cells in the TM and (G) TH+ fibres in the striatum from a 97% lesioned animal 3 days after 6-OHDA injection. (H) High-power photomicrograph of the boxed area in G. (I) A significant loss of TH cells was seen within the ventral diencephalon-mesencephalon 3 days post-lesioning (n=16 lesioned, n=14 sham, n=7 control; mean±s.d.; Student's t-test; ***, P<0.001). (J) Colocalisation of TUNEL+ and TH+ cells in the TM 3 days after lesioning. (K) Significantly more TUNEL+ cells and numerous TUNEL+ TH+ cells were observed in lesioned as compared with sham-lesioned animals. No double-positive cells were seen in sham-lesioned animals (mean±s.d.; Student's t-test; ***, P<0.001). (L') Schematic representation of the lesioning apparatus. (L'') The location of the burr hole and injection site (*), at the junction of the frontal and parietal bones. (M) A significantly higher percentage of pyknotic cells (ANOVA with Tukey post-hoc test; P<0.01) was seen 12 hours (5±1.2; n=7) and 24 hours (11.9±2.9; n=7) after lesioning as compared with shams (12 and 24 hour sham-lesioned animals pooled; 1.7±1.6; n=10). Note the increase in the percentage of pyknotic cells with increasing lesion size, and the increase in the percentage of pyknotic cells in the lesioned animals between 12 and 24 hours. (N,O) 6-OHDA injection leads to a significant loss of cells (DAPI), including neurons (NeuN), within the TM, and more specifically to the loss of DA markers [Nurr1, engrailed 1 (En1), TH]. n=3-7; mean±s.e.m.; Student's t-test; ***, P<0.001. Scale bars: 1 mm in A,C,E,G; 150 µm in B,F,J; 250 µm in D,H.

View larger version (32K): [in this window] [in a new window]   Fig. 2. Cellular and functional recovery after 6-OHDA injection. (A) Behavioural response following amphetamine challenge in non-operated controls (n=4), and in animals 3 days after sham (n=9) or 6-OHDA injection (n=21). Data represent the number of times per minute that the newt's tail crosses a line in a grid (mean±s.d.; Student's t-test; *, P<0.05; **, P<0.01). (B) Correlation of motor behaviour with lesion size 3 days after lesioning. The loss of TH+ cells strongly correlates (r2=0.83) with a decrease in locomotor activity in response to amphetamine challenge. Each data point represents 1 animal (for sham-lesioned animals, data represent mean±s.d.). (C) Time course of behavioural recovery as assessed by acute amphetamine challenge (lesion, n=19; sham, n=9). (D) Time course of TH+ cell regeneration following 6-OHDA injection. Sham, n=4-14 animals per group per time point (d3, n=4; d5, n=6, d7, n=5; d10, n=6; d17, n=5; d24, n=4; d30, n=14). Lesioned, n=5-18 animals per group per time point (d3, n=5; d5, n=6; d7, n=5; d10, n=6; d17, n=5; d24, n=6; d30, n=18). Mean±s.e.m.; Student's t-test; *, P<0.05; **, P<0.01. (E-G) Photomicrographs showing TH+ neurons in TM following (E) sham operation, (F) 3 days post-lesion and (G) 30 days after 6-OHDA injection. Scale bar: 150 µm.

View larger version (58K): [in this window] [in a new window]   Fig. 3. Neurogenesis of TH+ cells after 6-OHDA injection. (A) Time course of cellular proliferation within the ventral diencephalon and mesencephalon following sham- or 6-OHDA-lesioning. At days 7 and 10, a significant increase was seen in the number of BrdU+ cells in 6-OHDA-lesioned as compared with sham-lesioned animals. Control (grey line) represents the number (3.1±1.5) of BrdU+ cells in animals receiving no surgery/injection. Data represent mean±s.e.m.; n=3-11; Student's t-test; *, P<0.05. (B') Localisation of NeuN+ (neurons) and GFAP+ (ependymoglia) cells along the third ventricle. (B'') The cells lining the ventricle also express Sox2. (C') Significantly more BrdU+ cells were seen away from the ependymoglia layer at 13 days as compared with 6 hours after a single BrdU pulse (n=3 lesion; n=3 sham; mean±s.e.m.; Student's t-test; ***, P<0.001). (C'') Merged confocal image showing several BrdU+ GFAP+ cells lining the ventricle 6 hours after BrdU pulse. (C''') After a 13-day chase period, BrdU+ cells appear in neuronal layers. (D') Total number of BrdU+ TH+ cells following lesioning. Sham-lesioned animals pulsed days 0-30, n=11; lesioned animals pulsed days 0-30, n=8. Lesioned animals pulsed days 0-3, n=8. Lesioned animals pulsed days 4-23, n=8. Mean±s.e.m.; ANOVA with Tukey post-hoc test; ***, P<0.001. (D'') Confocal image of two BrdU+ TH+ neurons (arrows). (E) Total number of cells (DAPI) and neurons (NeuN) in the TM at 3 and 30 days after lesioning confirming that the number of cells returned to control levels by 30 days. (F') Behavioural response following amphetamine challenge at day 3, 8 and 14 after AraC treatment. AraC has no effect on the behaviour of control animals. However, it blocks all the behavioural recovery at 8 and 14 days normally seen in lesioned animals. (F'') AraC blocks all TH recovery at 8 days and significantly reduces recovery at 14 days in lesioned animals as compared with lesioned animals not treated with AraC (mean±s.e.m.; Student's t-test; ***, P<0.001). (F''') No new cells (BrdU+) were seen at 8 days in AraC-treated lesioned newts confirming the action of AraC (n=4-8 newts per group).

View larger version (54K): [in this window] [in a new window]   Fig. 4. De novo expression of DA determinants during DA regeneration in the newt. (A) Time course of Msx-1/2 expression in the ventral diencephalon and mesencephalon following lesioning. Note the significant reduction in Msx1/2-expressing cells at 24 hours and the significant increase at 7 days. Mean±s.e.m.; ANOVA with Tukey post-hoc test; *, P<0.05. (B') Msx1/2 expression in control animals and (B'') 24 hours after sham lesion and after (B''') 24 hours and (B'''') 7 days in 6-OHDA lesioned animals. (C') Number of Nurr1+ cells in the TM 3, 8 and 13 days after lesioning. (C'') Proportion of Nurr1+ to TH+ cells in the TM. Note that at 8 days there are significantly more Nurr1+ than TH+ cells compared with controls, indicating that maturation of DA neurons occurs along the normal developmental pathway. Mean±s.e.m.; ANOVA with Tukey post-hoc test; **, P<0.005; ***, P<0.001. (C''') An example of TH and Nurr1 double-positive cells in the TM of a control animal, illustrating the expression of Nurr1 in all TH+ neurons. Scale bars: 200 µm in A; 150 µm in B.

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