Semester

Fall

Date of Graduation

2018

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Not Listed

Committee Chair

George Spirou

Committee Co-Chair

Peter Mathers

Committee Member

Peter Mathers

Committee Member

Eric Tucker

Committee Member

Sergiy Yakovenko

Committee Member

Visvanathan Ramamurthy

Abstract

In the auditory brainstem, the connection between globular bushy cells of the anteroventral cochlear nucleus and principal cells (PCs) of the medial nucleus of the trapezoid body (MNTB) is created by one of the largest nerve terminals in the central nervous system, the calyx of Held (CH). The characteristics of the CH:MNTB connection—a short developmental period (48-72 hours), accessibility for recording from pre- and postsynaptic components, and clear monoinnervated end point—make this system an ideal model system for studying nervous system development. Model systems undergo stereotyped stages of development, including exuberant overinnervation, competition between terminals, and a refinement of innervation through removal of weak inputs. However, unlike other similar model systems (climbing fiber:Purkinje cell, retinal ganglion cells:dorsolateral geniculate nucleus), it has been a long-standing question whether the CH:MNTB system undergoes competition. We investigated the innervation state of PCs using the novel technique of segmentation and 3D reconstruction of PCs and their associated inputs across important developmental timepoints (postnatal days (P)2,3,4,6,9,30). This was accomplished by application of serial block-face scanning electron microscopy (SBEM), a method of serial section electron microscopy providing high spatial resolution (~4-10nm) and a high degree of alignment between images with very little section loss. Applying this technique, we show early exuberant innervation of PCs (P2), establish that competition is a common process, and pinpoint the 24-hour period from P3-P4 as a uniquely active day in CH growth during which terminal contact with PCs increases at a rate exceeding 200 µm2/day. Common morphological characteristics of the CH:MNTB connection also became qualitatively evident based on 3D reconstructions, particularly an eccentric PC nucleus and preference for polarized terminal growth. Based on these observations, we undertook a quantitative study of polarity in CH:MNTB development using our 3D reconstructions. The results of this investigation demonstrate a novel polarity in development of both the CH and PC; developing PCs are characterized by eccentrically placed nuclei that establish an “intrasomatic polarity” that persists through young adulthood (P30). This polarity appears to define a unique territory opposite of the nuclear location that is amenable to growth of the calyx, is enriched in dendrites, and is selectively enlarged as the principal cell matures to create glia-free surface area for innervation. To our knowledge, this is the first report of a polarity program in the coordinated pre- and postsynaptic development of a non-laminar brain region. Additionally, our findings have codified a progression of dendritic pruning in the maturation of principal cells that may influence and be influenced by the developmental state of the cell. Taken in totality, these results indicate a highly polarized, systemic competitive process in the MNTB during the development of the calyx of Held and suggests potential mediators of competition that deserve further study.

Embargo Reason

Publication Pending

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