Functional Refinement in the Projection from Ventral Cochlear Nucleus to Lateral Superior Olive Precedes Hearing Onset in Rat

Principal neurons of the lateral superior olive (LSO) compute the interaural intensity differences necessary for localizing high-frequency sounds. To perform this computation, the LSO requires precisely tuned, converging excitatory and inhibitory inputs that are driven by the two ears and that are matched for stimulus frequency. In rodents, the inhibitory inputs, which arise from the medial nucleus of the trapezoid body (MNTB), undergo extensive functional refinement during the first postnatal week. Similar functional refinement of the ascending excitatory pathway, which arises in the anteroventral cochlear nucleus (AVCN), has been assumed but has not been well studied. Using whole-cell voltage clamp in acute brainstem slices of neonatal rats, we examined developmental changes in input strength and pre- and post-synaptic properties of the VCN-LSO pathway. A key question was whether functional refinement in one of the two major input pathways might precede and then guide refinement in the opposite pathway. We find that elimination and strengthening of VCN inputs to the LSO occurs over a similar period to that seen for the ascending inhibitory (MNTB-LSO) pathway. During this period, the fractional contribution provided by NMDA receptors (NMDARs) declines while the contribution from AMPA receptors (AMPARs) increases. In the NMDAR-mediated response, GluN2B-containing NMDARs predominate in the first postnatal week and decline sharply thereafter. Finally, the progressive decrease in paired-pulse depression between birth and hearing onset allows these synapses to follow progressively higher frequencies. Our data are consistent with a model in which the excitatory and inhibitory projections to LSO are functionally refined in parallel during the first postnatal week, and they further suggest that GluN2B-containing NMDARs may mediate early refinement in the VCN-LSO pathway

Maturation of calcium-dependent GABA, glycine, and glutamate release in the glycinergic MNTB-LSO pathway.

The medial nucleus of the trapezoid body (MNTB) is a key nucleus in high-fidelity temporal processing that underlies sound localization in the auditory brainstem. While the glycinergic principal cells of the MNTB project to all primary nuclei of the superior olive, during development the projection from MNTB to the lateral superior olive (LSO) is of interest because this immature inhibitory projection is known to undergo tonotopic refinement during an early postnatal period, and because during this period individual MNTB terminals in the LSO transiently release glycine GABA and glutamate. Developmental changes in calcium-dependent release are understood to be required to allow various auditory nuclei to follow high frequency activity; however, little is known about maturation of calcium-dependent release in the MNTB-LSO pathway, which has been presumed to have less stringent requirements for high-fidelity temporal following. In acute brainstem slices of rats age postnatal day 1 to 15 we recorded whole-cell responses in LSO principal neurons to electrical stimulation in the MNTB in order to measure sensitivity to external calcium, the contribution of different voltage-gated calcium channel subtypes to vesicular release, and the maturation of these measures for both GABA/glycine and glutamate transmission. Our results establish that release of glutamate at MNTB-LSO synapses is calcium-dependent. Whereas no significant developmental changes were evident for glutamate release, GABA/glycine release underwent substantial changes over the first two postnatal weeks: soon after birth L-type, N-type, and P/Q-type voltage-gated calcium channels (VGCCs) together mediated release, but after hearing onset P/Q-type VGCCs predominated. Blockade of P/Q-type VGCCs reduced the estimated quantal number for GABA/gly and glutamate transmission at P5-8 and the frequency of evoked miniature glycinergic events at P12-15, without apparent effects on spontaneous release of neurotransmitter, supporting a model in which P/Q-type VGCCs are required for mature synchronous synaptic transmission, but not for spontaneous vesicle release

Spontaneous miniature events occur independent of P/Q activation.

<p><b>A</b>) Example miniature PSCs recorded in TTX, before and after ω-agatoxin IVA, in a P12 slice. <b>B</b>) Mean mPSC frequency (left) and amplitude (right) for each neuron recorded before and after P/Q block. N = 8 cells, P12–15. Cell in A shown in gray.</p

Proportional contribution of VGCC subtypes to GABA/glycine and glutamate transmission.

<p>(<b>A–B</b>) Representative examples showing how VGCC contribution to GABA/gly and glutamate neurotransmission was determined in (<b>A</b>) a P4 slice, and (<b>B</b>) a P10 slice. Average GABA/gly (red) and glutamate (green) current traces are shown below, and correspond to the colored points above for each component. (<b>C–D</b>) Proportional contribution of each VGCC to GABA/gly and glutamate components for the P4 slice shown in A (<b>C</b>), and for the P10 slice shown in B (<b>D</b>).</p

Paired pulse ratios for GABA/gly component.

<p>Paired-pulse ratios for the GABA/gly component for different external Ca⁺⁺ concentrations, stimulation frequencies, and postnatal age. Non-parametric 1-way ANOVA (Kruskal-Wallis test) across age revealed significant differences between young and older ages in 2 and 4 mM Ca⁺⁺ (Bonferroni's post hoc *<0.05, ?<0.005, ∼0.0005). The N for each age group is shown in parentheses.</p

Percent contribution of different VGCC subtypes to GABA/gly and glutamate components.

<p>Percent contribution of different VGCC subtypes to GABA/gly and glutamate components.</p

P/Q-type Ca⁺⁺ channels mediate evoked miniature events at older ages.

<p><b>A</b>) (Left) Example current trace collected after 100 Hz stimulation (P12 cell), showing reduction in sPSC frequency after application of the P/Q-type antagonist ω-agatoxin IVA. (Right) Superimposed sPSCs (150 events) in control and in ω-agatoxin IVA, for the neuron shown in left; average traces in black. <b>B</b>) Mean frequency, but not amplitude or rise time, of sPSCs was affected by P/Q-block with ω-agatoxin IVA. Black lines connect control and drug measurements from individual cells. Cell in A shown in gray.</p

Short-term plasticity is Ca⁺⁺-dependent for both GABA/gly and glutamate.

<p><b>A</b>) PPRs for GABA/gly neurotransmission shift from facilitating at low external Ca⁺⁺ (0.1 mM) to depressing at high external Ca⁺⁺ (4.0 mM) (P = 0.0003, F = 12.2, Friedman test, Dunn's post hoc P<0.005). <b>B</b>) PPRs for glutamate also shift from facilitating to depressing in a Ca⁺⁺-dependent manner (P = 0.02, F = 7.7, Friedman test, Dunn's post hoc P<0.05). Recordings from P3–5 slices.</p