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doi: 10.1242/10.1242/dev.00464

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Pitx3 is required for motor activity and for survival of a subset of midbrain dopaminergic neurons

Pepijn van den Munckhof1,*, Kelvin C. Luk2,*, Line Ste-Marie3, Jane Montgomery3, Pierre J. Blanchet4, Abbas F. Sadikot2,{dagger} and Jacques Drouin1,{dagger}

1 Unité de recherche en génétique moléculaire, Institut de recherches cliniques de Montréal (IRCM), Montréal, QC H2W 1R7, Canada
2 Cone Laboratory, Department of Neurology and Neurosurgery, Montréal Neurological Institute, McGill University, Montréal, QC H3A 2B4, Canada
3 Laboratoire de neurobiologie, Centre de recherche, CHUM Hôpital Notre-Dame, Université de Montréal, Montréal QC H2L 4M1, Canada
4 Centre de recherche, CHUM Hôpital Saint-Luc, Université de Montréal, Montréal QC H2X 3J4, Canada



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  		Fig. 1. The aphakia (ak) mice have no detectable Pitx3 and a markedly reduced midbrain dopaminergic system. (A-D) Adjacent coronal midbrain sections containing the SN in P50 wild-type (A,C) and ak (B,D) mice immunostained for TH (A,B) and Pitx3 (C,D). Rostrocaudal positions are indicated as millimeters relative to bregma in the lower right corner. (E) High-power view of the SN shown in C, highlighting the nuclear staining. By contrast, none of the weak background staining in D was nuclear. (F-K) Equivalent rostral-to-caudal coronal midbrain sections of P100 wild-type (F,H,J) and ak (G,I,K) mice immunostained for TH. (L) Stereological analysis of TH-positive cells of the left SN and VTA in wild-type and ak mice. The data are represented as the means±s.e.m. (n=4). (M) Density of Nissl-stained cell bodies in the left SN and VTA of wild-type and ak mice (n=4). A statistically significant decrease in TH-positive cell bodies and density of Nissl-stained cell bodies was detected in the SN and VTA of ak mice compared with controls (P<0.01, t-test). (N,O) Coronal sections through the right SN (N) and VTA (O) of a P50 wild-type mouse immunostained for TH (fluorescein-labeled, green) and Pitx3 (rhodamine-labeled, red) analyzed by confocal microscopy. Scale bars: in A, 125 µm for A-D; in F, 250 µm for F-K; in E, 30 µm for E; in N and O, 30 µm.

 


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  		Fig. 2. TH-positive MesDA neurons are lost primarily during fetal period for the SN and during postnatal period for VTA. (A) Plane of sections for analysis of TH-positive neurons in brain of E12.5 embryos. (B-G) Equivalent rostral-to-caudal sections through the midbrain of E12.5 wild-type (B,D,F) and ak (C,E,G) embryos immunostained for TH. (H,I) Coronal midbrain sections containing the SN and VTA in P1 wild-type (H) and ak (I) mice immunostained for TH. (J) Stereological analysis of TH-positive cells of the left SN and VTA in P1 wild-type and ak mice. The data are represented as the mean±s.e.m. (n=4). (K) Stereological analysis of TH-positive cells of the left SN and VTA in P21 wild-type and ak mice (n=4). A statistically significant decrease in TH-positive cell bodies was detected in the SN of P1 and P21 ak mice compared with controls (P<0.01, t-test) with no difference in the VTA. (L) Frequency of apoptotic cells revealed by TUNEL assay in the left SNc of P1 wild-type and ak mice (n=4). A statistically significant increase in the frequency of apoptotic cells was detected in the SNc of P1 ak mice compared with controls (P<0.05, t-test).

 


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  		Fig. 3. The ak mice have deficient striatal dopaminergic innervation. (A,B) Coronal sections through the left striatum of P100 wild-type (A) and ak (B) mice immunostained for TH. Rostrocaudal positions are indicated as millimeters relative to bregma in the lower right corner. Scale bar: 250 µm. (C,D) DA concentration in dorsal (C) and ventral (D) striatum of P130 wild-type and ak mice. The data are represented as the mean±s.e.m. (n=4). A statistically significant decrease in DA concentration was detected in the dorsal and ventral striatum of ak mice compared with controls (P<0.01, t-test).

 


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  		Fig. 4. The ak mice have impaired spontaneous locomotor activity. (A) Spontaneous ambulatory activity of mice recorded over 23 hours. The distance (cm) covered during each 1 hour period is shown for wild-type and ak mice. (B) Average distance covered per hour for wild-type and ak mice during daytime and night-time. (C) Average ambulatory time spent per hour for the recordings shown in E during daytime and night-time. (D) Spontaneous stereotypic movements of mice recorded over 23 hours. The stereotypic movements during each 1 hour period are shown for wild-type and ak mice. (E) Average numbers of stereotypic movements per hour for wild-type and ak mice during daytime and night-time. (F) Average time spent making stereotypic movements per hour for the recordings shown in H during daytime and night-time. (G) Resting time of mice recorded over 23 hours. The resting time during each 1 hour period is shown for wild-type and ak mice. (H) Average resting time per hour for wild-type and ak mice during daytime and night-time. (I) Average speed of spontaneous ambulatory movements for wild-type and ak mice during daytime and night-time. All locomotor activity data are represented as the mean ± s.e.m. (n=5). Locomotor activity scores for ak mice that are significantly different from wild-type scores are marked with a asterisk (P<0.01, t-test).

 


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  		Fig. 5. Similar distribution of MesDA neuronal losses in ak mice and in individuals with Parkinson's disease (PD). (A) The right midbrain of a normal mouse showing the distribution of TH-positive Pitx3-positive (green with red core) and TH-positive Pitx3-negative neurons (green) in SNc and VTA. Most ventral SNc TH-positive neurons are Pitx3 positive, whereas the dorsal SNc largely contains Pitx3-negative TH-positive neurons. About half of the VTA TH-positive neurons are Pitx3 positive, and both populations are intermingled. (B) In ak mice, SNc Pitx3-positive neurons are lost between E12.5 and P1, whereas VTA cells are lost postnatally. (C) Outline of the right MesDA system of a normal human showing the distribution of TH-positive neurons in SNc and VTA. (D) Individuals with PD typically have the most severe cell depletion in ventral SNc, followed by dorsal SNc and VTA [modified, with permission, from Jellinger (Jellinger, 2001Go)]. Although a decrease of PITX3-positive neurons was shown in samples from individuals with PD (Smidt et al., 1997Go), the regional distribution of human PITX3-positive neurons remains to be established.

 




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