Distribution of glial cells in the auditory brainstem: Normal development and effects of unilateral lesion
Introduction
Precise neural circuits in the auditory brainstem compute binaural timing and intensity disparities that are used to localize sound sources. In mammals, auditory information is carried by the VIIIth cranial nerve into the central nervous system (CNS), where branches of VIIIth nerve fibers terminate onto targets in the ventral cochlear nucleus (VCN). VCN globular bushy cells project to the contralateral medial nucleus of the trapezoid body (MNTB) where their large reticulated terminations, the calyces of Held, synapse onto principal neurons (Kuwabara et al., 1991, Kuwabara and Zook, 1991, Kandler and Friauf, 1993, Kil et al., 1995). MNTB neurons in turn provide glycinergic inhibition to the medial superior olive (MSO) and the lateral superior olive (LSO), which integrate excitation and inhibition to compute interaural time differences and interaural level differences, respectively.
This unique projection matures over a protracted period of development (Nakamura and Cramer, 2011). Axons reach contralateral MNTB and form immature connections by embryonic day (E) 17 (Borst and Soria van Hoeve, 2012). At postnatal day (P) 0, rudimentary calyces are seen with several inputs on each MNTB neuron. As the terminations expand, the number of VCN inputs is reduced until a single input encapsulates each MNTB neuron by P4 (Hoffpauir et al., 2006, Holcomb et al., 2013). Synapse formation and pruning have been shown to involve several forms of cell–cell communication. Notably, glial-secreted factors play a role in synaptic maturation (Mauch et al., 2001, Christopherson et al., 2005, Hughes et al., 2010, Kucukdereli et al., 2011, Allen et al., 2012, Korn et al., 2012). In addition, several types of glial cells have been shown to be important for synaptic refinement both in development and in response to injury (Chung and Barres, 2012, Karimi-Abdolrezaee and Billakanti, 2012, Schafer et al., 2012, Wake et al., 2013).
While the contribution of glial cells to the maturation of the central auditory circuitry is not known, recent studies have reported that astrocytes contact both the pre- and postsynaptic membranes of the calyx of Held (Elezgarai et al., 2001). These astrocytes elicit slow inward currents in the postsynaptic MNTB neuron via gliotransmission in the mature animal (Reyes-Haro et al., 2010). The close apposition of astrocytes to the calyx suggests a potential role for astrocytes in the development and function of this pathway.
To explore the role(s) of glial cells in the maturation of auditory circuits, we characterized the spatiotemporal emergence of glial subtypes in the VCN and MNTB. We used several markers to identify multiple astrocyte-specific proteins, including the intermediate filament glial fibrillary acidic protein (GFAP), the calcium binding protein S100β, and aldehyde dehydrogenase 1 family member L1 (ALDH1L1) (Cahoy et al., 2008). Oligodendrocytes were identified by expression of oligodendrocyte transcription factor 2 (OLIG2). The emergence of microglia was assessed by expression of the ionized calcium binding adaptor molecule 1 (IBA1).
Additional clues to mechanisms of neural circuit formation may be obtained from experimentally induced reorganization of synapses. Following early postnatal unilateral cochlear removal, the cochlear nucleus on the deafferented side undergoes substantial cell death (Trune, 1982, Hashisaki and Rubel, 1989, Mostafapour et al., 2000). Axons from the intact VCN subsequently branch and contact the ipsilateral, denervated MNTB, in addition to their normal contralateral target (Moore and Kowalchuk, 1988, Kitzes et al., 1995, Hsieh and Cramer, 2006, Hsieh et al., 2007). Here we examined the expression of glial markers in denervated and intact MNTB after cochlear removal.
During normal development we found a diversity of patterns for development of glial cell types in VCN and MNTB from birth to the time of hearing onset. We found that expression of astrocyte and oligodendrocyte markers following cochlear removal was similar to the distribution of these glial markers during normal development. In addition, glial cells and their processes were seen in close proximity to the emerging ipsilateral calyx; as in normal development, these populations were primarily astrocytes. Together with our developmental expression data, we posit that glial cells may be important for the development and early plasticity of the mammalian auditory circuit, and that different glial cell types may serve distinct functions in this pathway.
Section snippets
Animals
Wild-type mice on CD-1 background were used for these studies. Expression studies included animals at several developmental ages, including postnatal day (P)0 (n = 6); P6 (n = 9); P14 (n = 10); and P23 (n = 7). Cochlear removal (CR) or sham operation was performed at P2 and animals survived until P4 (n = 9 for CR and n = 9 for sham) or P9 (17 CR and 11 sham). All procedures were approved by the University of California, Irvine Institutional Animal Care and Use Committee.
Immunohistochemistry
Pups were euthanized with isoflurane
Expression of glial markers during the first postnatal week
As early as P0, glial markers were evident within the VCN, with notable variability in the expression of astrocyte proteins. GFAP at this age was sparse, limited to the ventral portion and in the central projection of the auditory nerve (Fig. 1A). ALDH1L1 (Fig. 1B) levels were abundant in the VCN, with less expression in the dorsal cochlear nucleus (DCN). S100ß displayed a pattern similar to that seen with GFAP (Fig. 1C).
The oligodendrocyte marker OLIG2 was expressed in the ventral portion of
Discussion
We have characterized the developmental emergence of distinct glial populations in the auditory brainstem, focusing on the projection from VCN to MNTB in relation to the developing calyx of Held. Additionally, we performed similar studies on lesion-induced ipsilateral projections. These studies provide clues to the function of glial cells in auditory synaptogenesis, and suggest that induced calyces share similar glial involvement with normal contralateral calyces.
Conclusions
Our data show distinct expression profiles of glial markers in the developing mouse brainstem. We found that glial cells emerge early in development, during synaptogenesis of the VCN–MNTB circuit, and that these populations shift and change overtime. The results are consistent with a role for glial cells and their associated proteins in synapse formation and maturation in the auditory brainstem. Following CR, we found that the expression of glial markers after surgery was largely similar to
Acknowledgements
The authors are grateful to H. Nguyen and J. Ayala Salazar for technical support and to Dr. S. Cohen-Cory and Dr. K. Green for assistance with imaging. This work was supported by NIH T32 DC010775.
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