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Cholinergic markers in the middle layers of rat auditory cortex are transiently upregulated during the second postnatal week, at which time alpha 7 nicotinic acetylcholine receptors (nAChRs) selectively regulate NMDA receptor (NMDAR)-mediated EPSPs. To investigate the developmental role of this regulation, we determined whether manipulating nAChR function at specific times during the first 4 weeks after birth could alter subsequent neuronal function. Rat pups were injected twice daily with nicotine (1 or 2 mg/kg) or saline during approximately the first, second, or fourth postnatal week (i. e., before, during, or after the peak upregulation of nAChRs). Glutamate EPSPs and intrinsic membrane properties were measured during whole-cell recordings from visually identified pyramidal neurons in layers II-IV of brain slices prepared at least 15 hr after the last injection. Chronic nicotine exposure (CNE) had little effect on intrinsic membrane properties and during week 1 or 4 did not affect synaptic function. However, CNE during week 2 resulted in EPSPs with long durations, multiple peaks, and enhanced NMDAR components. These changes remained significant even 10 d after CNE. Rapid application of nicotine, which in control neurons selectively enhances NMDAR EPSPs during week 2, produced only weak effects after CNE. Receptor binding studies showed that CNE-induced EPSP alterations occurred in the absence of altered alpha 7 nAChR numbers or agonist binding affinity. Thus, altered stimulation of nAChRs by CNE during week 2, but not before or after, disrupts the development of glutamate synapses in rat auditory cortex.

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.

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.