Ephrin-B reverse signaling is required for formation of strictly contralateral auditory brainstem pathways.

Specificity in the projections from the mammalian ventral cochlear nucleus (VCN) is essential for sound localization. Globular bushy cells project from the VCN to the medial nucleus of the trapezoid body (MNTB) on the contralateral, but not the ipsilateral, side of the brainstem, terminating in large synaptic endings known as calyces of Held. The precision in this pathway is critical for the computation of interaural intensity differences, which are used in sound localization. The mechanisms underlying the development of this projection are not completely understood. In this study, we tested the role of Eph receptor tyrosine kinases and their ephrin ligands in limiting the VCN-MNTB projection to the contralateral side. We found that mice with null mutations in EphB2 and EphB3 had normal contralateral VCN-MNTB projections, yet these projections also had significant numbers of aberrant collateral branches in the ipsilateral MNTB. These aberrant branches ended in calyceal terminations in MNTB. Similar ipsilateral projections were seen in mice with mutations in ephrin-B2. In both of these mouse lines, ipsilateral projections formed concurrently with normal contralateral projections and were not eliminated later in development. However, mice with mutations that affected only the intracellular domain of EphB2 had normal, strictly contralateral VCN-MNTB projections. Expression studies showed that EphB2 is expressed in VCN axons and ephrin-B2 is expressed in MNTB. Together, these data suggest that EphB2-ephrin-B2 reverse signaling is required to prevent the formation of ipsilateral VCN-MNTB projections and that this signaling operates non-cell autonomously.

Deletion of EphA4 enhances deafferentation-induced ipsilateral sprouting in auditory brainstem projections.

Axonal selection of ipsilateral and/or contralateral targets is essential for integrating bilateral sensory information and for coordinated movement. The molecular processes that determine ipsilateral and contralateral target choice are not fully understood. We examined this target selection in the developing auditory brainstem. Ventral cochlear nucleus (VCN) axons normally project to the medial nucleus of the trapezoid body (MNTB) only on the contralateral side. However, after unilateral removal of cochlear input in neonates, we found that axons from the unoperated VCN sprout and project to MNTB bilaterally. We found that EphA4 is expressed in the mouse auditory brainstem during development and during a sensitive period for ipsilateral sprouting, so we hypothesized that deletion of the Eph receptor EphA4 would impair target selection in these auditory pathways. Lipophilic dyes were used to evaluate quantitatively the brainstem projections in wild-type and EphA4-null mice. VCN-MNTB projections in EphA4-null mice were strictly contralateral, as in wild-type mice. However, after deafferentation, EphA4-null mice had a significant, threefold increase in the proportion of axons from the intact VCN that sprouted into ipsilateral MNTB compared with wild-type mice. Heterozygous mice had a twofold increase in these projections. These results demonstrate that EphA4 influences auditory brainstem circuitry selectively in response to deafferentation. Although this axon guidance molecule is not by itself necessary for appropriate target choice during normal development, it is a strong determinant of ipsilateral vs. contralateral target choice during deafferentation-induced plasticity.

Deafferentation induces novel axonal projections in the auditory brainstem after hearing onset.

Deafferentation of neural tissue can result in cell death, morphological changes, and/or alterations in sources of innervation. These changes often occur during a limited period of development. In the auditory brainstem, the ventral cochlear nucleus (VCN) projects to the contralateral but not ipsilateral medial nucleus of the trapezoid body (MNTB). This pathway is part of a circuit that computes interaural intensity differences used in sound localization. Previous studies have shown that, after the cochlea is removed early in postnatal development, cells in the VCN on the deafferented side die, and the intact VCN innervates MNTB on both sides of the brain. These changes after cochlea removal are limited to an early postnatal period that preceeds hearing onset. In this study, we lesioned the VCN directly to evaluate plasticity in axonal pathways after hearing onset. We found that novel projections from the intact VCN to ipsilateral MNTB emerge after lesions performed as late as postnatal day 25. The morphological sequence of events is similar to that seen during the initial development of this pathway. These data suggest that plasticity in the auditory brainstem is possible when pathways are challenged with denervation of target nuclei. The results show that the opportunity for plasticity in auditory brainstem circuitry is more prolonged than previously thought and that novel pathways can form after the normal pathways are fully mature and functional. Moreover, sensitive periods for changes in individual pathways are independently regulated.

Postnatal development of NR2A and NR2B mRNA expression in rat auditory cortex and thalamus.

Sensory cortex in the rat undergoes rapid postnatal development, especially following the onset of sensory function during so-called "critical periods." To investigate potential mechanisms in the auditory forebrain involving different NMDA receptor subunits, we have used in situ hybridization to determine expression patterns of NR2A and NR2B mRNA at postnatal days 4, 10, 13, 18, 25, and adult. In auditory cortex, NR2A mRNA expression is initially weak but increases rapidly over approximately 2 weeks. NR2B mRNA levels are initially high and remain high. For both subunits, expression tends to be highest in superficial layers of the cortex (except layer 1). Expression is weaker in the auditory thalamus (medial geniculate). Initially, NR2A mRNA expression is very low, whereas NR2B mRNA expression is moderate; both levels increase over approximately 2 weeks. Among medial geniculate subdivisions, NR2A mRNA expression occurs preferentially in the medial division, whereas NR2B mRNA expression is strongest in the ventral division. For auditory cortex and thalamus, NR2A and NR2B mRNA expression peaks about 1 week after the onset of hearing before declining slightly into adulthood. The heterogeneous distribution of NMDAR subunit mRNA throughout development may play a role in auditory forebrain development and function.

Nicotine exposure during a postnatal critical period alters NR2A and NR2B mRNA expression in rat auditory forebrain.

Chronic nicotine exposure (CNE) can alter brain development and is thought to produce deficits in auditory function. Previously, we found that CNE during the second postnatal week, but not before or after, increases the duration of excitatory postsynaptic potentials (EPSPs) mediated by N-methyl-D-aspartate receptors (NMDARs) in rat auditory cortex. It was proposed that a potential mechanism underlying increased EPSP duration could be over-stimulation of presynaptic nicotinic acetylcholine receptors, leading to prolonged glutamate release. Since glutamatergic activity regulates levels of postsynaptic NMDAR subunits, here we examine the effects of CNE on mRNA expression for the NR2A and NR2B subunits in auditory cortex and thalamus. Two days of CNE (postnatal days 8-9), produced no effects, but 5 days (postnatal days 8-12) enhanced cortical NR2A mRNA levels and reduced thalamic NR2B mRNA levels for up to 2 weeks. These effects are consistent with the hypothesis that CNE during a postnatal critical period disrupts auditory cortex development by over-stimulating glutamatergic synapses.

EphB signaling regulates target innervation in the developing and deafferented auditory brainstem.

Precision in auditory brainstem connectivity underlies sound localization. Cochlear activity is transmitted to the ventral cochlear nucleus (VCN) in the mammalian brainstem via the auditory nerve. VCN globular bushy cells project to the contralateral medial nucleus of the trapezoid body (MNTB), where specialized axons terminals, the calyces of Held, encapsulate MNTB principal neurons. The VCN-MNTB pathway is an essential component of the circuitry used to compute interaural intensity differences that are used for localizing sounds. When input from one ear is removed during early postnatal development, auditory brainstem circuitry displays robust anatomical plasticity. The molecular mechanisms that control the development of auditory brainstem circuitry and the developmental plasticity of these pathways are poorly understood. In this study we examined the role of EphB signaling in the development of the VCN-MNTB projection and in the reorganization of this pathway after unilateral deafferentation. We found that EphB2 and EphB3 reverse signaling are critical for the normal development of the projection from VCN to MNTB, but that successful circuit assembly most likely relies upon the coordinated function of many EphB proteins. We have also found that ephrin-B reverse signaling repels induced projections to the ipsilateral MNTB after unilateral deafferentation, suggesting that similar mechanisms regulate these two processes.

Regulation of glutamate synapses by nicotinic acetylcholine receptors in auditory cortex.

Acetylcholine plays an important role in regulating the processing of sensory stimuli, and understanding its specific cellular actions is critical to understanding how sensory cortex develops and functions in different behavioral states. Here we review recent work on the cellular effects of nicotinic receptor activation in auditory cortex and describe how these actions could affect systems-level auditory function. In particular, we describe a novel function of nicotinic acetylcholine receptors to regulate glutamate synapses containing N-methyl-D-aspartate receptors during early postnatal development. The transient regulation of developing glutamate synapses also defines a window of vulnerability during which exposure to exogenous nicotine disrupts synapse development. Thus, it appears that nicotinic regulation of glutamate synapses is a critical feature of auditory cortex development.