Dylan Haynes Mentor: Javier Fierro Washbourne Lab 8/17/2012.
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Dylan Haynes Mentor: Javier Fierro Washbourne Lab 8/17/2012 Functional Analysis of 4.1B-a During Synaptogenesis
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Transcript of Dylan Haynes Mentor: Javier Fierro Washbourne Lab 8/17/2012.
- Slide 1
- Dylan Haynes Mentor: Javier Fierro Washbourne Lab 8/17/2012
- Slide 2
- Synaptogenesis Washbourne et al. 2004 4.1B
- Slide 3
- Glutamate Glutamate Neurotransmitter Receptors Pre-Synaptic Terminal Post-Synaptic Terminal Glutamatergic Transmission in Cultured Neurons 4.1B
- Slide 4
- Danio rerio (Zebrafish) for In Vivo Studies of 4.1B 4.1B-a 4.1B-b 0.2 hpf 16 hpf 25 hpf Spontaneous Contraction Spontaneous Contraction Touch Evoked Response Touch Evoked Response
- Slide 5
- 4.1B-a is Important for Glutamatergic Transmission Necessary for the Touch-Response Neuronal Circuit Our Hypothesis
- Slide 6
- Experimental Design Exon 1 Exon 2 Exon 3Exon 4Exon 5Exon 6Exon 29 Morpholino Exon 1 Truncated Non-Functional Protein Exon 7
- Slide 7
- Experimental Design I1 RV Exon 1 Exon 2 Exon 3Exon 4Exon 5Exon 6Exon 29 Exon 7 E1 FW Intron Inclusion 250 bp Product
- Slide 8
- Experimental Design Exon 1Exon 2 Exon 3Exon 4Exon 5Exon 6Exon 29 E1 FW I1 RV Intron Exclusion No Product (Primer Cannot Bind) Exon 7
- Slide 9
- Experimental Design Exon 1 Exon 2 Exon 3Exon 4Exon 5Exon 6Exon 29 Exon 7 E7 RV E1 FW Intron Inclusion 550 bp
- Slide 10
- Experimental Design Exon 1Exon 2 Exon 3Exon 4Exon 5Exon 6Exon 29 Exon 7 E1 FW E7 RV Intron Exclusion 400 bp
- Slide 11
- Experimental Design I1 RV Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6 Exon 29 Exon 7 E1 FW Genomic DNA 250 bp Product (As Before)
- Slide 12
- Experimental Design E7 RV Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6 Exon 29 Exon 7 E1 FW Genomic DNA No Product (Too Big)
- Slide 13
- RT-PCR WT E-I WT E-E 0.4 mM E-I 0.4 mM E-E 0.6 mM E-I0.6 mM E-E 0.8 mM E-I0.8 mM E-E 1.0 mM E-I 1.0 mM E-E 1.2 mM E-I1.2 mM E-E Genomic E-I Genomic E-E 500 bp 100 bp 1000 bp Experimental Design Control Water Injected 0.8 mM 4.1B-a MO 1.2mM 4.1B-a MO Acridine Orange
- Slide 14
- IHC CaP VaP Colocalization Colocalization at CaP & VaP Pre-Synaptic Marker Synapsin 1,2 Post-Synaptic Marker Pan MAGUK p < 0.05 n = 6 n = 14 10 m
- Slide 15
- Touch Response Spinal Cord RB CoPA RDIN? PMN
- Slide 16
- Touch Response Control Response Morpholino Injected Response 40 ms 70 ms
- Slide 17
- Touch Response p < 0.02 n = 11 Control Morpholino Injected
- Slide 18
- 4.1B-a Knock-down: Reduces Excitatory Synapse Number in Primary Motor Neurons Reduces Velocity of Touch Evoked Tail Swing Increases Latency for Response to Touch 4.1B-a is Required for Proper Development of the Touch-Response Neuronal Circuit Conclusions
- Slide 19
- Rescue Experiments of 4.1B-a Overexpression Experiments of 4.1B-a Analyze: Synapse Number at CoPA Cells Spontaneous Tail Coiling Free Swimming Behavior at 3 dpf 4.1B-b Knock-Down Experiments Future Directions
- Slide 20
- Phil Washbourne Acknowledgments Daniel Gibson Spencer Larson Dan Fowler Renee Arias Francesco Argenton Domenico Cieri Yuqing Kou Raluca Chebac Neena Leggett Paola Pizzo
- Slide 21
- Special Thanks UO SPUR Peter ODay NICHD R25 Summer Research Program (NIH-1R25HD070817) Javier Fierro Jr.