High-fidelity transmission acquired via a developmental decrease in NMDA receptor expression at an auditory synapse

Abstract

Central auditory relay synapses in mature animals follow high-frequency inputs for computation of sound localization. In immature mice, however, transmission at the calyx of Held synapse in auditory brainstem was inaccurate for high-frequency inputs because the summed slow synaptic potential components caused aberrant firings or blocked action potentials. As the mice matured, synaptic potentials became shorter, with smaller and faster NMDA receptor components, thereby establishing the precise one-to-one transmission for high-frequency inputs. Developmental acquisition of this high-fidelity transmission could be mimicked experimentally in immature mice by blocking NMDA receptors with d(-)2-amino-5-phosphonovaleric acid (d-APV). Furthermore, bilateral cochlear ablations at postnatal day 7 (P7) attenuated the developmental decrease of NMDA receptor expression and prevented the acquisition of high-fidelity transmission. We suggest that auditory activity, which begins at P10-P12 in mice, downregulates the expression of postsynaptic NMDA receptors, thereby contributing to the establishment of high-fidelity synaptic transmission.

Fig. 1.

Postnatal development of high-fidelity synaptic transmission. A, Postsynaptic responses evoked by a train of high-frequency stimuli (21 pulses at 10, 20, 50, or 100 Hz) in mice at different postnatal periods (P7, P15, P27). Dotsand asterisks indicate the timing of stimuli and aberrant action potentials, respectively, in this figure and in Figure3. Resting potentials were −71 mV (P7), −69 mV (P15), and −72 mV (P27), respectively. B, Number of postsynaptic action potentials for 210 stimuli at 100 Hz at different postnatal periods. Of 27 cells, 16 cells showed a block and 8 cells showed aberrant firings at P7. Aberrant firings were found in 8 of 17 cells at P15 and none in 10 cells at P27. C, Developmental increase in the fidelity of transmission. “Fidelity” in the ordinate is defined as (210 − n_a)/210, wheren_a represents the number of aberrant spikes during 210 stimuli (for details, see Materials and Methods). Data points and error bars in this and the following figures indicate means ± SEM. Each data point was derived from 8–27 cells. In P7 mice the data of action potential block are excluded from this plot.

Fig. 2.

Pharmacological properties of EPSPs and EPSCs recorded from MNTB neurons. A, Action potentials triggered by EPSPs elicited by extracellular stimulation of an MNTB principal neuron at P7. GYKI 52466 (100 μm) abolished action potentials but did not block the slow EPSP component. This component was abolished by the additional application ofd-APV (50 μm). Averaged records before (Control) and during applications of GYKI and GYKI + d-APV are superimposed. The resting potential was −67 mV. B, Left, EPSCs evoked at the holding potential of −79 mV under voltage clamp in another MNTB neuron at P7. EPSCs were blocked by GYKI 52466 (100 μm; before and after GYKI records are superimposed). Right, In the presence of GYKI, EPSCs appeared as outward current at the holding potential of +51 mV. These EPSCs were abolished by d-APV (50 μm; superimposed).

Fig. 3.

Gain in fidelity of transmission by blocking NMDA receptors. A, Postsynaptic responses during a train of 100 Hz stimuli in MNTB neurons at different postnatal periods. In immature mice d-APV (50 μm) abolished aberrant firings and relieved action potentials from block, thereby making synaptic transmission accurate. Resting potentials were −69 mV (P7), −72 mV (P15), and −67 mV (P27). B, A similar effect of d-APV on postsynaptic responses at 35°C in a P7 mouse. Resting potential was −66 mV. C, Summary of the effects of d-APV on the fidelity of transmission at 100 Hz (n = 10–11) before (▴) and after (▵)d-APV application at 26–27°C. d-APV had no effects on synaptic fidelity in P27 mice.

Fig. 4.

Developmental changes in NMDA-EPSCs.A, Developmental decrease in the mean amplitude of NMDA-EPSCs recorded at +51 mV holding potential in the presence of CNQX (20 μm). Each data point was derived from 15–23 MNTB neurons. Sample traces are averaged NMDA-EPSCs (10 events) in P7, P13, and P27 mice, superimposed (top column), and shown with their peak amplitudes normalized and aligned at the stimulus artifact (bottom column).

Fig. 5.

Developmental changes in AMPA-EPSCs.A, Developmental increase in the mean amplitude of AMPA-EPSCs recorded at −79 mV holding potential (n= 15–23). After EPSCs were recorded, CNQX (20 μm) was applied, and the small CNQX-resistant component was subtracted for the data plotted in this graph. The superimposed sample traces are averaged EPSCs (10 events) at −79 mV holding potential in P7, P13, and P27 mice aligned at the stimulus artifact. Note that the synaptic latency is shorter at more mature mice. B, The 10–90% rise time and the decay time constant of AMPA-EPSCs at different postnatal age. The decay time could be fit adequately by a single exponential function for the data presented in the time plot. The superimposed sample traces are AMPA-EPSCs at P7, P13, and P27 normalized in amplitude and aligned at their peak.

Fig. 6.

Developmental changes in the expression of NMDA receptor subunit mRNAs and proteins. A, mRNAs encoding ζ1, ε1 and ε2, and G3PDH were detected by RT-PCR, using primers specific for ζ1, common to ε1 and ε2, or specific for G3PDH.B, Relative amounts of mRNA encoding ζ1 (●) and ε1/2 (▴) expressed at different postnatal days. Amounts of mRNA relative to G3PDH mRNA were measured and normalized to those at P5. Mean values were derived from three experiments. C, Immunoblots of ζ1, ε1, and ε2 subunit proteins at different postnatal days. D, Relative amounts of ζ1 (●), ε1 (■), and ε2 (▵) protein expressed at different postnatal days, deduced from immunoreactivity (from 3 experiments), and normalized to the values at P5.

Fig. 7.

Effects of bilateral cochlear ablation on synaptic fidelity and NMDA receptor expression. A, Percentage of mice showing positive ABR to 85 dB clicks. Inset records are ABRs from sham-operated (▴) and operated P13 mice (▵; bilateral cochlea ablated at P7) derived from the same litter. Numbers of untreated mice (○)were 10 for each period and 40 and 39, respectively, for sham-operated and operated mice. B, Synaptic fidelity during 100 Hz stimulation in operated and sham-operated control mice at P14–P16 (n = 10 mice each). C, Mean amplitude of NMDA-EPSCs in P14–P16 mice (n = 10). D, Amounts of ε1/2 mRNA (left) and ζ1 mRNA (right) expressed in the MNTB region in sham-operated and operated mice at P14–P16. Ordinates indicate the amount of mRNA encoding NMDA receptor subunits relative to G3PDH mRNA. Data measured from the same experiments (using 5 mice each) are connected with lines (4 experiments). Difference was significant (p < 0.02, paired t test) for ε1/2 mRNAs but was not significant for ζ1 mRNAs (p > 0.1).