Semester

Spring

Date of Graduation

2009

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Biochemistry

Committee Chair

Peter H. Mathers.

Abstract

Developing neurons utilize multiple guidance cues to reach their appropriate destination. Although much is known about the anatomy and electrophysiology of auditory brainstem neurons, the molecular factors directing migration of these cells and the targeting of their axons are only beginning to be explored. Ventral cochlear nucleus (VCN) neurons have axons that project bilaterally to the superior olivary complex (SOC) in the ventral acoustic stria (VAS). The circumferential trajectory taken by developing VCN axons is similar to the path of spinal commissural neurons (SCNs). Therefore, we reasoned that netrin-DCC and slit-robo signaling systems may function, similar to SCNs, in the guidance of VCN axons. We found that VCN axons extend toward the midline as early as E13, with many axons crossing the midline by E14.5. During this interval, netrin-1 and slit-1 RNAs are expressed at the brainstem midline, VCN neurons express RNAs for DCC, robo-1 and robo-2 receptors and VCN axons in the VAS are immunoreactive for DCC. Additionally, VCN axons fail to reach the midline in netrin-1- or DCC-deficient mice. The lack of VCN axonal outgrowth in DCC-deficient mice is not explained by the modest reduction (∼ 10%) of VCN neurons. Taken together, these data show that a functional netrin-DCC signaling system is required for establishing proper VCN axonal projections in the auditory brainstem.;During our characterization of VCN axonal projections, we found that the neurons comprising the medial nucleus of the trapezoid body (MNTB), a component of the SOC, are absent in mice carrying no functional netrin-1 or DCC alleles. Since DCC is known to direct cellular migration in other regions of the central nervous system, we hypothesized that netrin-DCC and slit-robo signaling systems may function in the migration and positioning of MNTB neurons. RNAs for DCC, robo-1 and robo-2 are expressed in columns of cells that extend from the rostral brainstem to the developing SOC. MNTB and other superior olivary complex (SOC) neurons maintain expression of these guidance receptors during the formation of discreate SOC nuclei. Additionally, mice carrying only one functional copy of the netrin-1 or DCC allele have a laterally displaced MNTB and express half the quantity of DCC found in wild-type animals. The displaced MNTB is maintained in adult animals carrying only one functional DCC allele and is not explained by a difference in the number of MNTB neurons, as determined by unbiased stereology. In addition, the trochlear nucleus and ventral tegmental nucleus are significantly displaced, medially and laterally, respectively, in these adult heterozygous animals. We found that the tonotopic organization of the laterally displaced MNTB is preserved in mice carrying only one functional DCC allele. Therefore, these auditory circuits have sufficient plasticity to properly form in the presence of a significantly displaced MNTB. We propose that netrin-DCC and slit-robo signaling is necessary for the migration of SOC neurons and that medial-to-lateral positioning of the SOC and other central nervous system nuclei occurs in a dose-dependent manner, based on a cell's programmed response to midline signals. This work will have clinical implications for future research in the repair/regeneration of central nervous system circuits.

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