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Progress report

Progress report. 28.03.2014 Niels Ntamati. VTA projection neurons. Dopaminergic efferents. GABAergic efferents. Fields et al., 2007. Optogenetic targeting. GAD 65 -Cre mouse. VTA. Tan et al., 2012. VTA-GABA fibers in the Hippocampus. Dentate Gyrus. DG. CA3. gcl – granule cell layer

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Progress report

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  1. Progress report 28.03.2014 NielsNtamati

  2. VTA projection neurons Dopaminergic efferents GABAergic efferents Fields et al., 2007

  3. Optogenetic targeting GAD65-Cre mouse VTA Tan et al., 2012

  4. VTA-GABA fibers in the Hippocampus

  5. Dentate Gyrus DG CA3 gcl – granule cell layer ml – molecular layer pcl – pyramidal cell layer pl – polymorphic layer (hilus) Amaral et al., 2007

  6. Retrograde labeling of VTA input CTB merge CTB • injection of cholera toxin subunit B – Alexa555 (CTB) in the DG (WT mouse) • 2 weeks incubation DAPI

  7. Retrograde labeling of VTA input merge • injection of cholera toxin subunit B – Alexa555 (CTB) in the DG (WT mouse) • 2 weeks incubation • anti-TH immunostaining merge TH TH A M L DAPI DAPI P CTB ipn CTB

  8. Retrograde labeling of VTA input • injection of AAV5-DIO-EYFP in VTA (GAD-Cre mouse) • injection of cholera toxin subunit B – Alexa555 (CTB) in the DG • 3 weeks incubation • anti-TH immunostaining EYFP merge eYFP CTB TH CTB

  9. Retrograde labeling of VTA input • injection of AAV5-DIO-EYFP in VTA (GAD-Cre mouse) • injection of cholera toxin subunit B – Alexa555 (CTB) in the DG • 3 weeks incubation • anti-TH immunostaining merge merge A EYFP M L P EYFP TH TH CTB ipn 100 uM CTB

  10. VTA-DG connectivity GFP merge DAPI

  11. VTA-DG connectivity • MCL internal (Ecl = -5 mV) • Vc = -70 mV • 2 light pulses (4ms, IPI = 50 ms) 10 pA 10 ms

  12. VTA-DG connectivity ≥ 3 x SD of baseline noise 40/57 cells light-responsive non-responsive

  13. Target cell excitability light-resp non-resp 12 non-resp, light OFF non-resp, light ON light-resp, light OFF 2 +400 pA light-resp, light OFF -200 -> 80 pA 4 4 Acc Reg -200 pA 200 pA 400 pA regular accomodating

  14. Identification of the target cell biocytin in recording pipette GFP / DAPI / biocytin

  15. VGLUT expression in the DG DAPI VGLUT1 merge H GCL ML

  16. VGLUT expression in the DG DAPI VGLUT2 merge H GCL ML

  17. VGLUT expression in the DG DAPI VGLUT2 biocytin merge ChR2-EYFP

  18. Prox1 Prox1 DAPI merge ML GCL H

  19. Prox1 DAPI PROX1 biocytin merge ChR2-EYFP

  20. Identification of the target cell biocytin in recording pipette GFP / DAPI / biocytin Amaral et al., 2007

  21. Polysynaptic connection? TTX + 4-AP Petreanu et al., 2009 20 pA 10 ms

  22. Mixed GABA/Glu currents

  23. Mixed GABA/Glu currents P<0.001 p=0.049

  24. Mixed GABA/Glu currents

  25. Mixed GABA/Glu currents p=0.006 p=0.048

  26. Cs+ methane-sulfonate recordings • CsMS internal (Ecl = -47 mV, Ena,K = 0 mV) • Vc = -50 / 0 mV • 2 light pulses (4ms, IPI = 50 ms)

  27. Cs+ methane-sulfonate recordings

  28. Cs+ methane-sulfonate recordings 10 pA 10 ms p=0.029

  29. Cs+ methane-sulfonaterecordings 10 pA 10 ms

  30. Cs+ methane-sulfonaterecordings

  31. Whatnext? • VGAT-Cre confirmation

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