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Dylan Haynes Mentor: Javier Fierro Washbourne Lab 8/17/2012

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Synaptogenesis Washbourne et al. 2004 4.1B

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Glutamate Glutamate Neurotransmitter Receptors Pre-Synaptic Terminal Post-Synaptic Terminal Glutamatergic Transmission in Cultured Neurons 4.1B

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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

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4.1B-a is Important for Glutamatergic Transmission Necessary for the Touch-Response Neuronal Circuit Our Hypothesis

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Experimental Design Exon 1 Exon 2 Exon 3Exon 4Exon 5Exon 6Exon 29 Morpholino Exon 1 Truncated Non-Functional Protein Exon 7

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Experimental Design I1 RV Exon 1 Exon 2 Exon 3Exon 4Exon 5Exon 6Exon 29 Exon 7 E1 FW Intron Inclusion 250 bp Product

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Experimental Design Exon 1Exon 2 Exon 3Exon 4Exon 5Exon 6Exon 29 E1 FW I1 RV Intron Exclusion No Product (Primer Cannot Bind) Exon 7

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Experimental Design Exon 1 Exon 2 Exon 3Exon 4Exon 5Exon 6Exon 29 Exon 7 E7 RV E1 FW Intron Inclusion 550 bp

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Experimental Design Exon 1Exon 2 Exon 3Exon 4Exon 5Exon 6Exon 29 Exon 7 E1 FW E7 RV Intron Exclusion 400 bp

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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)

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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)

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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

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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

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Touch Response Spinal Cord RB CoPA RDIN? PMN

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Touch Response Control Response Morpholino Injected Response 40 ms 70 ms

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Touch Response p < 0.02 n = 11 Control Morpholino Injected

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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

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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

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Phil Washbourne Acknowledgments Daniel Gibson Spencer Larson Dan Fowler Renee Arias Francesco Argenton Domenico Cieri Yuqing Kou Raluca Chebac Neena Leggett Paola Pizzo

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Special Thanks UO SPUR Peter ODay NICHD R25 Summer Research Program (NIH-1R25HD070817) Javier Fierro Jr.