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

Synaptogenesis Washbourne et al B

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

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

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

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

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

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

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

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

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)

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)

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

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 = µm

Touch Response Spinal Cord RB CoPA RDIN? PMN

Touch Response Control Response Morpholino Injected Response 40 ms 70 ms

Touch Response p < 0.02 n = 11 Control Morpholino Injected

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

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

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

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