1. Hindbrain Neurons

2. Question: How will the alteration of input affect neurons in the CNS dedicated to inner ear sensory input processing? By manipulating the ear, either by removing it or by adding extra ears, we can determine the effects of afferents on their target neuron(s) in the CNS

3. Background Ear ablation results in the reduction in the volume and number of neurons in the cochlear nuclei (Levi- Montalcini; Rubel) Early removal of the ear results in neuronal loss, whereas later removal results in virtually no cell death in the cochlear nuclei of postnatal animals Indicates a critical period of cochlear nucleus neuron dependence on afferent input, possibly including activity (Rubel) (Levi-Montalcini, 1949)

4. Background Previous studies have focused on whole neuronal populations It was thought that some neurons were more susceptible to death during the critical period than others, possibly related to ‘birth-dates’. Whether a critical period exists for a given neuron has never been determined http://www.pic2fly.com/Neuron+3D+Model.html ?

5. Mauthner Cell Single, large, pair of reticulospinal neurons at the level of the ear in the hindbrain of most aquatic animals Important component of the C-start escape response 25µm

6. Mauthner Cell Early, but not late, ear ablation has been shown to result in the absence of the Mauthner cell Has been suggested to be a surgical artifact Surviving Mauthner cells show a reduction in dendritic branching Previous studies each only focused on a single time point of ear removal I removed the ear at various time points in development to answer the following questions:

7. Mauthner Cell Questions 1) Is there a critical period of a Mauthner cell during which it depends on the ear for survival? 2) Does the stage at which the ear is removed affect the degree of dendritic branching of surviving Mauthner cells?

8. Methods Ears were removed at various stages of Xenopus laevis development Otic placode (Stg 24-26) For some, ears were replaced Early otic vesicle (Stg 27-30) Later otic vesicle (Stg 31-40)

9. Methods Ears were removed at various stages of Xenopus laevis development Otic placode (Stg 24-26) For some, ears were replaced Early otic vesicle (Stg 27-30) Later otic vesicle (Stg 31-40) Embryos allowed to grow until stage 46

10. Mauthner Cell Survival Control Remove and Replace Ear (n = 29) Hindbrain Dextran amine dye application

11. Mauthner Cell Survival Dextran amine dye3A10 Antibody Ear Removed 50µm

12. Mauthner Cell Survival 100% Mauthner cell survival in stages 36-40 embryos 100% Mauthner cell survival in remove-replace % 38%64%95% N= 91N= 39N= 40

13. Questions 1) Is there a critical period of a Mauthner cell during which it depends on the ear for survival? 2) Does the stage at which the ear is removed affect the degree of dendritic branching of surviving Mauthner cells?

14. Dendritic Branching following ear loss ControlRemove and Replace Ear Ear Removed at Placode (Stage 26)Ear Removed at Late Otic Vesicle (Stage 40) 25µm 23 Terminal Branches26 Terminal Branches19 Terminal Branches24 Terminal Branches 3 Terminal Branches32 Terminal Branches9 Terminal Branches

15. Increase Afferent Input: Extra Ears Donor ear from stage 25-27 Xenopus embryo is transplanted to host, rostral to native ear

16. Dendritic Branching with extra ear 50µm Extra Ear 50µm 21 Terminal Branches33 Terminal Branches

17. Dendritic Branching *** p < 0.001 n = 12 *** * n = 10 Control Treated * p < 0.05 ControlRemove-ReplaceRemove Placode (24-26) Remove Early Otic Vesicle (27-30) Remove Late Otic Vesicle (27-30) Extra Ear t-test

18. How will the Alteration of Input Affect Neurons in the CNS Dedicated to Inner Ear Sensory Input? The earlier the ear is removed, the less likely the Mauthner cell will survive. Critical period appears to be mostly over at Stage 31 Is unlikely to be a surgical artifact (remove and replace experiment) Ear removal results in significantly reduced dendritic branching There is a trend that the later the ear is removed: reduction in dendritic branching is less severe Degree of dendritic branching may be related to the relative number of afferent neurons projecting into the hindbrain, but may have an upper limit