The Role of Spontaneous Activity in Maturation of the Calyx of Held Nerve Terminal and Its Synaptic Target in the Medial Nucleus of the Trapezoid Body

Graduation Year

2024

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medical Engineering

Major Professor

George Spirou, Ph.D.

Committee Member

Henrique von Gersdorff, Ph.D.

Committee Member

Joseph Walton, Ph.D.

Committee Member

Huabei Jiang, Ph.D.

Committee Member

Nathan Gallant, Ph.D.

Keywords

Astrocyte, Auditory Brainstem, Development, Electrophysiology, Neurotransmission

Abstract

Neural circuit formation is a complex process involving synergistic roles between neurons and glial cells regulated by coordinated genetic and molecular cues and activity-dependent mechanisms. The canonical pathway for synapse formation begins with neurogenesis and migration followed by axonal outgrowth and dendritic arborization allowing for the capture and formation of nascent synapses. These initial synaptic contacts are exuberant in nature ensuring proper neural connectivity and undergo a period of pruning of superfluous inputs followed by maturation and strengthening of the remaining contacts. This maturation includes pre- and postsynaptic specialization of the active zone, localization and clustering of ion channels, and myelination regulating conduction timing. Initial synapse formation can occur independent of neural activity, but the process of maturation and strengthening is an activity-dependent mechanism. Interestingly, in sensory systems, such as the visual and auditory system, this complex process of synaptic pruning and strengthening occurs prior to the onset of external stimulation (in mice, ear canals and eyes open after postnatal day (P)10). However, Intrinsic patterned spontaneous activity (SA) occurs in the sensory end organ and percolates through connected brain regions and plays a role in the topographical organization of sensory systems through activity-dependent refinement and maturation. The calyx of Held (CH) is the primary terminus of globular bushy cells (GBCs), whose cell bodies are located in the ventral cochlear nucleus (VCN) and innervates principal neurons (PNs) in the medial nucleus of the trapezoid body (MNTB). The MNTB was used as a model system for studying neural circuit formation and maturation, in part because the CH nerve terminal grows rapidly between P2 and P4 and MNTB PNs are mostly refined to mono-innervation within the first postnatal week, concurrent with glial cell expansion. Additionally, the biophysical properties of the CH:MNTB synaptic partners have been well characterized.

An under-studied maturational step in this system is the onset and progression for myelination of the calyx-forming (calyceal) and MNTB PN axons. We investigated the onset and progression of developmental myelination in the MNTB utilizing two complementary large volume datasets, assaying the temporal dynamics of myelin gene expression and ultrastructure of the myelin sheath during the first two postnatal weeks. We utilized a developmental time series of microarrays to analyze significantly changing genes during the second postnatal week (P6-P14) in the MNTB. Comparison to a published cell-type specific RNA-seq data set from the cortex showed enrichment of most upregulated genes in oligodendrocyte lineage (OL) cells. Furthermore, gene ontology analysis identified the most enriched categories associated with OL maturation and myelination. To determine the temporal dynamics of myelin gene expression we performed quantitative polymerase chain reaction qPCR on a subset of genes related to myelination and found a continuum for the pattern of activation instead of distinct waves. Given the prevalence of OL cell and myelination related gene expression we utilized our developmental series of serial block-face scanning electron microscopy (SBEM) image volumes to assay the onset and progression of myelination in the MNTB at ultrastructural resolution. Utilizing a combination of ultrastructural morphological criteria from the literature and our own SBEM volumes we classified glia cells in the MNTB allowing identification and assignment of individual glial processes to a specific cell-type, showing prominent astrocyte wrapping of calyceal axons prior to myelination by OL cells. We identified a novel feature of astrocytes in the MNTB, where transient astrocytic ensheathment of calyceal axons could be priming and targeting axons for subsequent OL cell wrapping and myelination.

To test the requirement of SA for CH growth and synchronized pre- and postsynaptic maturation we utilized a viral vector approach, expressing tetanus neurotoxin (TeNT), to selectively block synaptic transmission at the CH:MNTB connection. TeNT is expressed under control of the pUNISHER cassette, which allows for rapid-onset and high levels of expression. Unilateral viral injections were performed at P0 targeting the VCN with detectable fluorescence (mCherry co-expressed with TeNT) in the contralateral MNTB as early as P2. The efficacy of viral vector mediated synaptic silencing was assayed through whole-cell patch-clamp recordings with a significant reduction in the frequency of spontaneous excitatory postsynaptic currents compared to ipsilateral controls. Furthermore, paired recordings simultaneously patching the CH and MNTB PNs showed abolished synaptic transmission following presynaptic depolarizing current injections. Blocking activity at the CH:MNTB connection resulted in MNTB PNs remaining in an immature hyperexcitable state, continuing into the second postnatal week. Segmentation and 3D reconstruction of calyces from both the ipsilateral and contralateral MNTB show a reduced volume and increased thickness of transduced calyces at P6, and P9. This study highlights an important role for SA triggering rapid growth of the CH and the synchronous maturation of the MNTB PN physiological properties.

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