Structure-function relation of the developing calyx of Held synapsein vivo

Abstract

In adult rodents, a principal neuron in the medial nucleus of the trapezoid (MNTB) is generally contacted by a single, giant axosomatic terminal called the calyx of Held. How this one-on-one relation is established is still unknown, but anatomical evidence suggests that during development principal neurons are innervated by multiple calyces, which may indicate calyceal competition. However, in vivo electrophysiological recordings from principal neurons indicated that only a single strong synaptic connection forms per cell. To test whether a mismatch exists between synaptic strength and terminal size, we compared the strength of synaptic inputs with the morphology of the synaptic terminals. In vivo whole-cell recordings of the MNTB neurons from newborn Wistar rats of either sex were made while stimulating their afferent axons, allowing us to identify multiple inputs. The strength of the strongest input increased to calyceal levels in a few days across cells, while the strength of the second strongest input was stable. The recorded cells were subsequently immunolabeled for vesicular glutamate transporters (VGluT) to reveal axosomatic terminals with structured-illumination microscopy. Synaptic strength of the strongest input was correlated with the contact area of the largest VGluT cluster at the soma (r = 0.8), and no indication of a mismatch between structure and strength was observed. Together, our data agree with a developmental scheme in which one input strengthens and becomes the calyx of Held, but not with multi-calyceal competition.Key points summaryDuring development the giant, auditory calyx of Held forms a one-to-one connection with a principal neuron of the medial nucleus of the trapezoid body.While anatomical studies described that most of the target cells are temporarily contacted by multiple calyces, multi-calyceal innervation was only sporadically observed in in vivo recordings, suggesting a structure-function discrepancy.We correlated synaptic strength of inputs, identified in in vivo recordings, with post hoc labeling of the recorded neuron and synaptic terminals containing vesicular glutamate transporters (VGluT).During development only one input increased to the level of the calyx of Held synapse, and its strength correlated with the large VGluT cluster contacting the postsynaptic soma.As neither competing strong inputs nor multiple large VGluT clusters on a single cell were observed, our findings did not indicate a structure-function discrepancy.

Volume electron microscopy reveals age-related circuit remodeling in the auditory brainstem

“…It has been extensively described that presynaptic CH terminals make multiple small contacts on MNTB neurons during the early developmental critical period (first postnatal week), followed by an early stage of functional and structural transformation (Kandler and Friauf, 1993;Taschenberger et al, 2002;Wimmer et al, 2006;Rodríguez-Contreras et al, 2008). During this pruning, multiple inputs strengthen and compete until a final single innervation is established (Holcomb et al, 2013;Sierksma et al, 2020). Recently, it was reported that the MNTB can still display multiple CH inputs in young mice even after the critical period (3 W and 4 W; Milinkeviciute et al, 2019Milinkeviciute et al, , 2021.…”

“…During development, MNTB neurons receive multiple synaptic inputs from the globular bushy cells located in the cochlear nucleus. Already before hearing onset (around P12-P14), just one giant calyx terminal contacts one postsynaptic cell, after a process of elimination of multiple small inputs, pruning and strengthening of the "winner" terminal (Kuwabara et al, 1991;Hoffpauir et al, 2006;Holcomb et al, 2013;Sierksma et al, 2020). In some pathological conditions like the deletion of bone morphogenetic protein (Xiao et al, 2013), alteration in the microglia (Milinkeviciute et al, 2019(Milinkeviciute et al, , 2021 or potentiation of the medial olivocochlear system (Di Guilmi et al, 2019), the presence of multiple calyces on a principal MNTB has been reported beyond the critical period.…”

“…This temporary developmental gradient along the tonotopic axis is established during the critical period before the hearing onset (P12-P14). Considering that calyceal competition occurs during the same developmental windows (Rodríguez-Contreras et al, 2008;Sierksma et al, 2020) and that the maturation of neurotransmission progresses from high to low frequencies along the tonotopic axis (Yu and Wang, 2022), one can speculate that the only plastic window for eliminating terminals is during the critical period. Since low frequencies mature later, one can consider that not all MNTB cells located in this region mature before the end of the critical period, leaving an incomplete elimination of its calyceal inputs.…”