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

Abstract

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.

Figure 1. Short-term plasticity is Ca⁺⁺-dependent for both GABA/gly and glutamate.

A) 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) 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.

Figure 2. Paired-pulse properties of GABA/gly, but not glutamate, component are significantly altered by developmental age.

A) Representative GABA/glycine-mediated PSCs (average of 10 traces) recorded in the presence of CNQX (5 µM). B) Glutamate-mediated PSCs (average of 10 traces) recorded in the presence of picrotoxin (50 µM), strychnine (10 µM), and cyclothiazide (100 µM). All recordings, in response to 100 Hz electrical stimulation in the MNTB, at a holding potential of −70 mV, with an external Ca⁺⁺ concentration of 2 mM. C–E) Summary of PPRs at inter-pulse intervals of 10, 20, 50, and 100 ms for the GABA/gly component in (C) 0.1 mM (D) 2.0 mM, and (E) 4.0 mM external Ca⁺⁺. * P<0.05 (Kruskal-Wallis test; Dunn's post hoc). F–H) Summary of PPRs for glutamate component at 10, 20, 50, and 100 ms inter-pulse intervals in (F) 0.1 mM, (G) 2.0 mM, and (H) 4.0 mM external Ca⁺⁺. Open symbols in C–E indicate which groups showed significant differences.

Figure 3. Proportional contribution of VGCC subtypes to GABA/glycine and glutamate transmission.

(A–B) Representative examples showing how VGCC contribution to GABA/gly and glutamate neurotransmission was determined in (A) a P4 slice, and (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. (C–D) Proportional contribution of each VGCC to GABA/gly and glutamate components for the P4 slice shown in A (C), and for the P10 slice shown in B (D).

Figure 4. Proportional contribution of VGCC subtypes to neurotransmission between P3 and P15.

A) Percent contribution of L, N and P/Q-type VGCCs to GABA/gly transmission P3–15 (N for each age group in parentheses). The three VGCCs made significantly different contributions at different ages ([2,123] = 14.76, P<0.0001, 2-way ANOVA; Bonferroni post-tests, * P<0.05, ** P<0.005, *** P<0.0005). B) Percent contribution of L, N and P/Q-type VGCCs to glutamate transmission P3–10. L and N-types made similar contributions, while P/Q-type made significantly larger contributions at later ages ([2, 99] = 60.77, P<0.001, 2-way ANOVA; Bonferroni post-tests P<0.05). For ages P3–10, both a GABA/gly and a glutamate component were recorded from each neuron.

Figure 5. P/Q-type VGCCs influence quantal release for GABA/gly and glutamate component.

A) PSC traces in control and with ω-agatoxin IVA. GABA/gly and glutamate traces were recorded from the same neuron (P6). GABA/gly traces below from P13 tissue. Each trace shown is the average of 10 responses. B–C) Estimated quantal number for GABA/gly component (B) and glutamate component (C) before and after application of ω-agatoxin IVA at P5–8, calculated from the pooled data on the left and from individual cells on the right. The last 8 data points from the curve were fitted by linear regression and extrapolated to time zero to estimate the readily releasable pool. The cumulative amplitude found by the linear regression was divided by the average amplitude of the sPSC (for GABA/gly 29.0±6.1 pA in control and 26.8±4.7 pA after ω-agatoxin IVA; for glutamate 21.4±2.6 pA in control and 20.0±2.9 pA after ω-agatoxin IVA. Cell in A shown in gray.

Figure 6. P/Q-type Ca⁺⁺ channels mediate evoked miniature events at older ages.

A) (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) 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.

Figure 7. Spontaneous miniature events occur independent of P/Q activation.

A) Example miniature PSCs recorded in TTX, before and after ω-agatoxin IVA, in a P12 slice. 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.