Investigation of Spatial Effects on Dopamine Reuptake Mechanism - PowerPoint Presentation

1. Investigation of Spatial Effects on Dopamine Reuptake MechanismCihan Kaya12/15/2015

2. AimsBuild and simulate a spatial model of dopaminergic signaling.Investigate the effects of Spatial distribution or surface density fluctuations of dopamine transportersGeometry

3. Difference between dopaminergic and glutamatergic synapsesPropertyDopamineGlutamaterefsTransporter localizationDA axons, somata and dendritesPre- and postsynaptic, surrounding gliaNirenberg et al., 1996aSeal and Amara, 1999Danbolt, 2001Transporter cycle rate (molecules/s/transporter)2–535Wadiche et al., 1995Povlock and Schenk, 1997Prasad and Amara, 2001Intersynaptic distance (forebrain)1.2–3.5 μm0.5 μmDoucet et al., 1986Pickel et al., 1981Descarries et al., 1996Cragg and Rice, 2004Arbuthnott and Wickens, 2007Receptor localizationextrasynapticintra- and extrasynapticSesack et al., 1994Yung et al., 1995Hersch et al., 1995Khan et al., 1998Ottersen and Landsend, 1997Galvan et al., 2006Receptor sensitivitynM-μMμM-mMRichfield et al., 1989Neve and Neve, 1997Receptor response time1–5 ms50 ms - >1 sRusakov and Kullman, 1998Barbour, 2001

4. Model Rice et. al., 2008.

5. Dopamine reuptake mechanismaDopamine clearance mediated with two mechanisms:Release: Content of a vesicle released instantly with a frequency. Uptake: Transporters will clear the EC DA.Membrane

6. aOldest Model for DA transmission [DA] : extracellular DA concentration[DA]p : DA concentration of a vesiclef : frequency of vesicular releaseVmax : Michelis-Menten max rate, function of DAT concentrationRelease termIssues with release termConstant increase in DA concentrationRelease frequency is constantUptake termIssues with uptake termConstant Vmax , should be function of [DAT]General ConcernsWell-mixed assumptionNo spatial informationGarris et. al., 1994.

7. Volume Transmission ModelTwo parameter for spatial variablesSingle synapse model with single releaseRice et. al., 2004.  ⍺: void fractionλ: tortuosityU: total volumeCf: # of DA in vesiclesk’: DA uptake rate  Gaussian diffusionFirst order decayGeneral ConcernsOversimplification of space and heterogeneityFirst order reaction mechanism for transporters

8. Simple Spatial ModelDATDAT-DADA (extracellular)DA (intracellular)No reference and no justificationRest of the parameters are from Michaelis-Menten kinetics.Rooney et. al., 2015.General ConcernsIntroduction of microscopic diffusion with very simple geometry. Uncertainties and lack of validation of parametersParameterValueNumber of dopamine molecules per vesicle1900Extracellular volume associated with a release site (dorsal striatum)2 × 10−15 lNumber of DAT associated with a release site2800DAT catalytic rate9 s−1DAT-DA binding on rate (transfected cells)5× 10+6 M−1 s−1DAT-DA binding on rate (tissue prep)1.3× 10+8 M−1 s−1DAT-DA binding off rate0.5 s−1Dopamine diffusion rate4 × 10−6 cm−2 s−1

9. New ModelNo well-mixed assumptionMicroscopic diffusion in the form of Monte Carlo diffusion of DA molecules is implemented. Image data is utilized to build geometry and place molecules.

10. Protocol3D GeometryReaction NetworkMCellOutput Trajectories10Garris et. al., 1994Cardona et. al., 2012

11. Image Data10x10 μm stacks with 0.4 μm separation. (18 images)Mouse striatum (HA-DAT strain) slicesStained with anti-HA antibodies and Cy3 anti-mouse antibodies. Red colors are dopamine transporters.Block et. al., 20155 μProjection (Superposition) of all slices

12. Manual 3D reconstructionDopamine transporters are highly expressed on membrane. Red color is used as membrane stain. Image reconstructed in ImageJ. Visualized in Blender. First SliceTop view

13. EnvironmentRemaining portion of the space is filled with other cells which do not have dopamine transporters. The gap between the DAT containing axons and environment is around 20 nm thick. (Block et. al, 2015)

14. Full reconstructed imageTOP VIEWISOMETRIC VIEW

15. DAT placement according to images13Large intensity regions have larger DAT density and rest of the axons have a lower density.Density numbers are used from Block et. al., 2015.

16. Active zone placement 3456Varicosities are determined from images. The region with low DAT intensity on the varicosity is selected as active zone.

17. Kinetics and Simulation SetupDATOUTDATOUT-DADA (extracellular)DATINDA (intracellular)ParameterValueNumber of dopamine molecules per vesicle650DAT surface density55 / μ2 and 115 / μ2DAT catalytic rate9 s−1DAT-DA binding onrate (transfected cells)5× 10+6 M−1 s−1DAT-DA binding offrate0.5 s−1Dopamine diffusion rate4 × 10−6 cm−2 s−1DATi-DATo transfer rate20 s-1DA firing rate1 HzTime step1 μInitially, all DAT are in outward facing state and simulation started with first firing of DA.

18. Results

19. Results

20. Outputs and analysesVolume (fL)Volume(fL)Site 1 – 1 μ0.196Site 2 – 1 μ0.187Site 1 – 2 μ1.610Site 2 – 2 μ1.190Site 1 – 5 μ20.459Site 2 – 5 μ22.790Site 1 – 10 μ83.881Site 2 – 10 μ91.600

21. Sample ResultsDue to oversimplification of geometry in volume transmission model, concentration of DA is smaller. To reach the concentration depicted from volume transmission model, there should be half DA molecules inside volume.

22. ConclusionThe spatial complexity has a significant effect on DAergic signaling. Image data is utilized to investigate spatiotemporal dynamics of DAergic signaling. Previous imaging efforts to investigate DAT transport is useful for predicting EC DA dynamics.

23. Future workThe model will be simulated with high resolution images (EM). The correlation between the distribution of DA and DAT is investigated. Alternative kinetics such as blocking of DAT transport will be investigated.