Dynamic development of the calyx of Held synapse

Abstract

The calyx of Held is probably the largest synaptic terminal in the brain, forming a unique one-to-one connection in the auditory ventral brainstem. During early development, calyces have many collaterals, whose function is unknown. Using electrophysiological recordings and fast-calcium imaging in brain slices, we demonstrate that these collaterals are involved in synaptic transmission. We show evidence that the collaterals are pruned and that the pruning already begins 1 week before the onset of hearing. Using two-photon microscopy to image the calyx of Held in neonate rats, we report evidence that both axons and nascent calyces are structurally dynamic, showing the formation, elimination, extension, or retraction of up to 65% of their collaterals within 1 hour. The observed dynamic behavior of axons may add flexibility in the choice of postsynaptic partners and thereby contribute to ensuring that each principal cell eventually is contacted by a single calyx of Held.auditory system ͉ medial nucleus of the trapezoid body ͉ two-photon imaging ͉ structural plasticity ͉ axon collateral S tudying the formation of individual, identified synapses in the CNS presents a formidable challenge because of their small size, their incredibly high density, and their protracted formation period (1). Imaging studies in living animals have provided insights into the structural changes that presynaptic axons undergo during development, which complements our understanding of how specific brain connections form (2). An emerging view from these studies is that axonal dynamics are age-and cell type-dependent (3) and strongly correlated with the formation of synaptic contacts, which may ultimately guide the growth of the axonal arbor (2, 4-6).Here, we study the development of a CNS synapse that can be identified relatively easily because it is probably the largest synaptic contact in the mammalian brain (7). The calyx of Held connects the globular bushy cells of the anteroventral cochlear nucleus and the principal cells of the medial nucleus of the trapezoid body (MNTB) in the brainstem. Studies in rodents have shown that shortly before birth, the principal cells of the MNTB are innervated by small glutamatergic boutons (8). Morphological and functional identification of nascent calyces is possible between postnatal days 3 and 5 (8-10), which suggests that the characteristic one-to-one innervation observed in mature animals is achieved very rapidly (10). However, previous studies have not been able to study the dynamic aspects of the calyx of Held development.As a first step toward elucidating the cellular mechanisms that ensure that each MNTB principal cell is always innervated by only a single calyx of Held, we sought an imaging approach. Because it has not been possible to study this unique synapse in culture, we carried out studies in vivo. We developed a surgical procedure to label brainstem axons in anesthetized rat pups and imaged them with a two-photon microscope. Using this approach, we provide evidence of structural dynamics r...

Synaptic Vesicles in Mature Calyx of Held Synapses Sense Higher Nanodomain Calcium Concentrations during Action Potential-Evoked Glutamate Release

“…In young calyces of Held, vesicular glutamate release showed a surprisingly high sensitivity to [Ca 2ϩ ] i : estimates for the peak [Ca 2ϩ ] i at vesicle fusion sites during APs (9 -28 M) (Bollmann et al, 2000;Schneggenburger and Neher, 2000) are substantially lower than those near the mouth of open VGCCs (Llinás et al, 1992;Yamada and Zucker, 1992;Roberts, 1994;Naraghi and Neher, 1997). However, the former estimates for the local [Ca 2ϩ ] i sensed by synaptic vesicles were obtained from P8 -P10 calyces corresponding to a developmental stage just 5-7 d after the formation of calyx synapses takes place on the majority of principal cells (Kandler and Friauf, 1993;Hoffpauir et al, 2006;Rodríguez-Contreras et al, 2008). Numerous studies suggest that not only morphological features but also functional properties of young calyx synapses are remarkably different from mature ones [see von Gersdorff and Borst (2002) and references therein].…”

“…During its postnatal maturation, the calyx synapse undergoes a multitude of morphological and functional transformations: the terminal is remodeled from an immature spoon-like morphology to the multidigit morphology seen in the adult (Kandler and Friauf, 1993), and calyceal collaterals gradually disappear (Rodríguez-Contreras et al, 2008). EPSCs are larger in older rats (Taschenberger and von Gersdorff, 2000;Iwasaki and Takahashi, 2001) and mice (Joshi and Wang, 2002), and the kinetics of APevoked release is substantially faster in mature calyces , rivaling and exceeding that measured at endbulb synapses (Isaacson and Walmsley, 1995).…”

Resistance to action potential depression of a rat axon terminal in vivo

“…The in vivo electroporation with Alexa Fluor 594-dextran (Molecular Probes) was described in ref. 51. The in vivo electrophysiological methods are detailed in ref.…”

“…The day of birth was taken as P0. Neonate pups were anesthetized with isoflurane, intubated, and mechanically ventilated, and underwent a ventral approach to expose the right ventral brainstem (22,51). Before the recordings, anesthesia was reduced to 1% isoflurane, which kept the animal areflexive.…”

Natural and lesion‐induced decrease in cell proliferation in the medial nucleus of the trapezoid body during hearing development

“…3B;Rodr ıguez-Contreras et al, 2008;Ford et al, 2009). A 403/NA1.4 oil lens attached to an LSM510 confocal microscope (Carl Zeiss) was used to acquire z-stacks of 8-bit, 512 3 512 pixel images.…”