Nigrostriatal Dopamine Terminals in DYT1 Dystonia: a 3D Ultrastructural Analysis in DYT1 Knock-in Mice Öffentlichkeit

Abstract

Importance: This thesis presents results of the very first ultrastructural study of the potential pre-synaptic morphological changes of dopamine terminals in the DYT1 KI mice. We used cutting-edge 3D-Electron Microscopy (EM) reconstruction approach to provide a comprehensive view of neural connections and potential structural changes of dopamine synapses in the striatum of DYT1 KI mice.

Objective: Because the underlying mechanism of the decreased dopamine (DA)release in the striatum of DYT1 mice remains unclear, we propose to study potential ultrastructural changes of nigrostriatal DA synapses that could contribute to the reduced striatal DA neurotransmission in DYT1 KI mice.

Materials and Methods: We did immunohistochemistry on dorsal striatal tissues from the WT and DYT1 KI mice groups. We took EM images of DA terminals labeled via tyrosine hydroxylase (TH) under Single Block Facing/Scanning Electron Microscopic (SBF/SEM). Using the Reconstruct software, we reconstructed 80 TH-immunoreactive (TH-IR) terminals in 2 WT and 2 DYT1 KI mice from which we collected the following data: (1) volume of DA terminals, (2) number and area of post-synaptic densities (PSDs) of DA synapses, (3) number and volume of mitochondria in DA terminals. We also took EM images of DA terminals labeled via dopamine transporter (DAT) immunostaining under Transmission Electron Microscope (TEM). We used images from 7 DAT-immunoreactive (DAT-IR) terminals in WT and in DYT1 KI to measure the surface area of vesicles in DAT-IR DA terminals using ImageJsoftware. 

Results: In DYT1 KI mice, (1) while the number and area of PSDs on axo-spinous synapses decreased (t-test, p=0.00561), the volume of post-synaptic spines increased (t-test, p= 0.0075). Also, (2) the number of mitochondria inside TH-IR terminals increased and was correlated with increased volume of TH-IR terminals. Additionally, we found that (3) the number of axo-axonic appositions increased, and (4) the spine-invaginated DA terminals decreased. Other changes in the volume of DA terminals, number and area of PSDs on axo-spinous synapses, volume of mitochondria, area of vesicles in DA terminals were found, but did not reach statistical significance.

Conclusion and Relevance:Our findings highlight altered axo-spinous synapses and post-synaptic spine morphology, increased number of mitochondria in pre-synaptic DA terminals, and increased incidence of axo-axonic dopamine appositions in the striatum of DYT1 dystonic mice models. These changes might contribute to impaired DA neurotransmission and modulation of other striatal afferents, or serve as compensation for the reduced DA release in the striatum of DYT1 mutant mice.

Table of Contents

1 Abstract............................................................................................................................................................................ 1

2 Background and Rationale ............................................................................................................................................. 3

2.1 DYT1 Dystonia and DYT1 Dystonic Mice Models.......................................................................................... 3

2.2 Reduced Dopamine (DA) Release in DYT1 Mice............................................................................................ 4

2.3 Rationale............................................................................................................................................................ 8

3 Hypothesis........................................................................................................................................................................ 9

4 Material and Methods....................................................................................................................................................... 9

4.1 Animals............................................................................................................................................................. 9

4.2 Antibodies......................................................................................................................................................... 9

4.3 TH- and DAT-Immunocytochemistry Protocol.............................................................................................. 10

4.4 Transmission Electronic Microscopy (TEM).................................................................................................. 10

4.5 Single Block Facing/Scanning Electron Microscopic (SBF/SEM)................................................................ 11

4.6 Ultrastructural 3D Reconstruction (Reconstruct)............................................................................................ 11

4.7 Neurotransmitter Vesicle Area (ImageJ)......................................................................................................... 12

4.8 Statistical analysis........................................................................................................................................... 12

5 Results............................................................................................................................................................................ 12

5.1 Changes in TH-IR terminal sizes between WT and DYT1 KI mice............................................................... 12

5.2 Morphological changes of TH-IR terminals in DYT1 KI mice...................................................................... 13

5.3 Striatal TH-IR synapses in WT vs DYT1 mice............................................................................................... 14

5.4 Changes in the Number of Mitochondria in TH-IR Terminals of DYT1 mice............................................... 15

5.5 Synaptic vesicles analysis: no difference in the size of synaptic vesicles in DAT-IR terminals between WT and DYT1 KI mice   16

6 Discussion...................................................................................................................................................................... 16

6.1 Morphological changes of TH-IR terminals might indicate compensation for reduced DA release.............. 18

6.2 Decreased number and area of PSDs on axo-spinous synapses might be associated with reduced DA release20

6.3 Increased number of axo-axonic appositions might indicate striatal afferents interplay and modulation of DA release      21

6.4 Increased number of TH-IR terminals containing more mitochondria might indicate active pre-synaptic mechanism24

6.5 No changes in the size of vesicles indicates normal neurotransmitter concentration and quantal size.......... 26

6.6 Increased post-synaptic spine volume might serve as a rescue for reduced DA neurotransmission.............. 27

6.7 Spine-invaginated terminals indicate more signal and remodeling of neurotransmission in the striatum...... 28

7 Conclusion...................................................................................................................................................................... 30

8 Tables and Figures.......................................................................................................................................................... 31

Table 1: Antibodies...................................................................................................................................................... 9

Table 2: Number of TH-IR terminals analyzed in each animal................................................................................. 13

Figure 1: TH-IR terminals in the striatum of WT and KI mice................................................................................. 31

Figure 2: Axo-dendritic and axo-spine synapses of striatal dopaminergic terminals................................................ 32

Figure 3: Quantitative analysis of the TH-IR terminals and their synaptic contacts................................................. 33

Figure 4: Mitochondria in the TH-IR terminals......................................................................................................... 34

Figure 5: Quantitative analysis of mitochondria........................................................................................................ 35

Figure 6: Analysis of the vesicle size in the DAT-immunoreactive terminals.......................................................... 36

Figure 7: Axo-axonic contacts of TH-IR terminals................................................................................................... 37

9 References...................................................................................................................................................................... 38

About this Honors Thesis

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School	Emory College
Department	Neuroscience and Behavioral Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Neuroscience
Stichwort	Ultrastructure Dystonia DYT1-Knock-in-Mice DAT-Immunostaining 3D-Reconstruction Striatum Dopaminergic-synapses TH-Immunostaining
Committee Chair / Thesis Advisor	Yoland Smith, Emory University
Committee Members	Leah Roesch, Emory University Rosa Villalba, Emory University Ellen Hess, Emory University

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