Adrián Rodríguez-Contreras

Calcium action potentials in hair cells pattern auditory neuron activity before hearing onset

We found rat central auditory neurons to fire action potentials in a precise sequence of mini-bursts before the age of hearing onset. This stereotyped pattern was initiated by hair cells in the cochlea, which trigger brief bursts of action potentials in auditory neurons each time they fire a Ca2+ spike. By generating theta-like activity, hair cells may limit the influence of synaptic depression in developing auditory circuits and promote consolidation of synapses.

Direct measurement of single-channel Ca2+ currents in bullfrog hair cells reveals two distinct channel subtypes

1 To confer their acute sensitivity to mechanical stimuli, hair cells employ Ca2+ ions to mediate sharp electrical tuning and neurotransmitter release. We examined the diversity and properties of voltage‐gated Ca2+ channels in bullfrog saccular hair cells by means of perforated and cell‐attached patch‐clamp techniques. Whole‐cell Ca2+ current records provided hints that hair cells express L‐type as well as dihydropyridine‐insensitive Ca2+ currents. 2 Single Ca2+ channel records confirmed the presence of L‐type channels, and a distinct Ca2+ channel, which has sensitivity towards ω‐conotoxin GVIA. Despite its sensitivity towards ω‐conotoxin GVIA, the non‐L‐type channel cannot necessarily be considered as an N‐type channel because of its distinct voltage‐dependent gating properties. 3 Using 65 mm Ca2+ as the charge carrier, the L‐type channels were recruited at about –40 mV and showed a single‐channel conductance of 13 pS. Under similar recording conditions, the non‐L‐type channels were activated at ∼–60 mV and had a single‐channel conductance of ∼16 pS. 4 The non‐L‐type channel exhibited at least two fast open time constants (τo= 0.2 and 5 ms). In contrast, the L‐type channels showed long openings (τo=∼23 ms) that were enhanced by Bay K 8644, in addition to the brief openings (τo= 0.3 and 10 ms). 5 The number of functional channels observed in patches of similar sizes suggests that Ca2+ channels are expressed singly, in low‐density clusters (2–15 channels) and in high‐density clusters (20–80 channels). Co‐localization of the two channel subtypes was observed in patches containing low‐density clusters, but was rare in patches containing high‐density clusters. 6 Finally, we confirmed the existence of two distinct Ca2+ channel subtypes by using immunoblot and immunohistochemical techniques.

Dynamic development of the calyx of Held synapse.

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.

Ca2+ transport properties and determinants of anomalous mole fraction effects of single voltage-gated Ca2+ channels in hair cells from bullfrog saccule

We studied the permeation properties of two distinct single voltage‐gated Ca2+ channels in bullfrog saccular hair cells to assess the roles of the channels as physiological Ca2+ transporters and multi‐ion pores. By varying the permeant ions (Ba2+, Ca2+) and concentrations (2–70 mm), we estimated the affinity constant (KD) of the two channels as follows (mm): L‐type channel, KD,Ba= 7.4 ± 1.0, KD,Ca= 7.1 ± 2.2 (n= 7); non‐L‐type channel, KD,Ba= 5.3 ± 3.2, KD,Ca= 2.0 ± 1.0 (n= 8). Using ionic concentrations close to physiological conditions (2 mm Ca2+ and 1.0 mm Mg2+), the conductance of the L‐type channel was ∼2 pS. We determined the mechanisms by which ions traverse the pore of these single Ca2+ channels, using mixtures of Ba2+ and Ca2+ at total concentrations above (70 mm) or close to (5 mm) the KD of the channels. We found evidence for an anomalous mole fraction effect (AMFE) only when the total divalent ion concentration was 5 mm, consistent with a multi‐ion pore. We show that AMFE arises from the boundaries between the pore and bulk solution in the atria of the channel, which is derived from the presence of depletion zones that become apparent at low divalent cation concentrations. The present findings provide an explanation as to why previous whole‐cell Ca2+ currents that were recorded in quasi‐physiological Ca2+ concentrations (∼2–5 mm) showed clear AMFE, whereas single Ca2+ channel currents that were recorded routinely at high Ca2+ concentrations (20–110 mm) did not.

Learning Drives Differential Clustering of Axodendritic Contacts in the Barn Owl Auditory System

Computational models predict that experience-driven clustering of coactive synapses is a mechanism for information storage. This prediction has remained untested, because it is difficult to approach through time-lapse analysis. Here, we exploit a unique feature of the barn owl auditory localization pathway that permits retrospective analysis of prelearned and postlearned circuitry: owls reared wearing prismatic spectacles develop an adaptive microcircuit that coexists with the native one but can be analyzed independently based on topographic location. To visualize the clustering of axodendritic contacts (potential synapses) within these zones, coactive axons were labeled by focal injection of fluorescent tracer and their target dendrites labeled with an antibody directed against CaMKII (calcium/calmodulin-dependent protein kinase type II, α subunit). Using high-resolution confocal imaging, we measured the distance from each contact to its nearest neighbor on the same branch of dendrite. We found that the distribution of intercontact distances for the adaptive zone was shifted dramatically toward smaller values compared with distributions for either the maladaptive zone of the same animals or the adaptive zone of normal juveniles, which indicates that a dynamic clustering of contacts had occurred. Moreover, clustering in the normal zone was greater in normal juveniles than in prism-adapted owls, indicative of declustering. These data demonstrate that clustering is bidirectionally adjustable and tuned by behaviorally relevant experience. The microanatomical configurations in all zones of both experimental groups matched the functional circuit strengths that were assessed by in vivo electrophysiological mapping. Thus, the observed changes in clustering are appropriately positioned to contribute to the adaptive strengthening and weakening of auditory-driven responses.

Branching of calyceal afferents during postnatal development in the rat auditory brainstem

Cells in the anteroventral cochlear nucleus (aVCN) send out calyceal axons that form large excitatory somatic terminals, the calyces of Held, onto principal cells of the contralateral medial nucleus of the trapezoid body (MNTB). It is unclear which fraction of these axons might form more than one calyx and whether this fraction changes during development. We combined in vitro anterograde tracing, stereological cell counts, analysis of apoptosis, and immunohistochemistry to study the development of calyceal afferents in rats of different postnatal ages. We found that some principal cells were contacted by multiple large axosomatic inputs, but these invariably originated from the same axon. Conversely, at least 18% of traced afferents branched to form multiple calyces, independently of age. Calyces from the same axon generally innervated nearby principal cells, and most of these branch points were <50 μm away from the synaptic terminals. Our results show that the projection from the aVCN to the MNTB is divergent, both when calyces have just been formed and in the adult. Cell counts did not provide evidence for principal cell loss during development, although analysis of apoptosis showed a large increase in nonneuronal cell death around the onset of hearing. Our data suggest that, once a calyceal synapse forms in the MNTB, it stays. J. Comp. Neurol. 496:214–228, 2006.

Effects of Permeant Ion Concentrations on the Gating of L-Type Ca2+ Channels in Hair Cells

We determined the gating and permeation properties of single L-type Ca(2+) channels, using hair cells and varying concentrations (5-70 mM) of the charge carriers Ba(2+) and Ca(2+). The channels showed distinct gating modes with high- and low-open probability. The half-activation voltage (V(1/2)) shifted in the hyperpolarizing direction from high to low permeant ion concentrations consistent with charge screening effects. However, the differences in the slope of the voltage shifts (in VM(-1)) between Ca(2+) (0.23) and Ba(2+) (0.13), suggest that channel-ion interaction may also contribute to the gating of the channel. We examined the effect of mixtures of Ba(2+) and Ca(2+) on the activation curve. In 5 mM Ca(2+), the V(1/2) was, -26.4 +/- 2.0 mV compared to Ba(2+), -34.7 +/- 2.9 mV, as the charge carrier. However, addition of 1 mM Ba(2+) in 4 mM Ca(2+), a molar ratio, which yielded an anomalous-mole fraction effect, was sufficient to shift the V(1/2) to -34.7 +/- 1.5 mV. Although Ca(2+)-dependent inactivation of the L-type channels in hair cells can yield the present findings, we provide evidence that the anomalous gating of the channel may stem from the closed interaction between ion permeation and gating.

Developmental Changes in Short-Term Plasticity at the Rat Calyx of Held Synapse

The calyx of Held synapse of the medial nucleus of the trapezoid body functions as a relay synapse in the auditory brainstem. In vivo recordings have shown that this synapse displays low release probability and that the average size of synaptic potentials does not depend on recent history. We used a ventral approach to make in vivo extracellular recordings from the calyx of Held synapse in rats aged postnatal day 4 (P4) to P29 to study the developmental changes that allow this synapse to function as a relay. Between P4 and P8, we observed evidence for the presence of large short-term depression, which was counteracted by short-term facilitation at short intervals. Major changes occurred in the last few days before the onset of hearing for air-borne sounds, which happened at P13. The bursting pattern changed into a primary-like pattern, the amount of depression and facilitation decreased strongly, and the decay of facilitation became much faster. Whereas short-term plasticity was the most important cause of variability in the size of the synaptic potentials in immature animals, its role became minor around hearing onset and afterward. Similar developmental changes were observed during stimulation experiments both in brain slices and in vivo following cochlear ablation. Our data suggest that the strong reduction in release probability and the speedup of the decay of synaptic facilitation that happen just before hearing onset are important events in the transformation of the calyx of Held synapse into an auditory relay synapse.

Axodendritic contacts onto calcium/calmodulin-dependent protein kinase type II-expressing neurons in the barn owl auditory space map.

In the owl midbrain, a map of auditory space is synthesized in the inferior colliculus (IC) and conveyed to the optic tectum (OT). Ascending auditory information courses through these structures via topographic axonal projections. Little is known about the molecular composition of projection neurons or their postsynaptic targets. To visualize axodendritic contacts between identified cell types, we used double-label immunohistochemistry, in vivo retrograde tracing, in vitro anterograde tracing, high-resolution confocal microscopy, three-dimensional reconstruction and fly-through visualization. We discovered a major class of IC neurons that strongly expressed calcium/calmodulin-dependent protein kinase type II, α subunit (CaMKII). The distribution of these cells within the IC was mostly restricted to the external nucleus of the IC (ICX), in which the auditory space map is assembled. A large proportion of ICX-OT projection neurons were CaMKII positive. In addition to being the principal outputs, CaMKII cells were in direct contact with axonal boutons emanating from the main source of input to ICX, the lateral shell of the central nucleus of the inferior colliculus (ICCls). Numerous sites of putative synaptic contact were found on the somata, proximal dendrites, and distal dendrites. Double-label immunoelectron microscopy confirmed the existence of synapses between ICCls axons and the dendrites of CaMKII cells. Collectively, our data indicate that CaMKII ICX neurons are a cellular locus for the computation of auditory space-specific responses. Because the ICCls-ICX projection is physically altered during experience-dependent plasticity, these results lay the groundwork for probing microanatomical rearrangements that may underlie plasticity and learning.

Release and Elementary Mechanisms of Nitric Oxide in Hair Cells

The enzyme nitric oxide (NO) synthase, that produces the signaling molecule NO, has been identified in several cell types in the inner ear. However, it is unclear whether a measurable quantity of NO is released in the inner ear to confer specific functions. Indeed, the functional significance of NO and the elementary cellular mechanism thereof are most uncertain. Here, we demonstrate that the sensory epithelia of the frog saccule release NO and explore its release mechanisms by using self-referencing NO-selective electrodes. Additionally, we investigated the functional effects of NO on electrical properties of hair cells and determined their underlying cellular mechanism. We show detectable amounts of NO are released by hair cells (>50 nM). Furthermore, a hair-cell efferent modulator acetylcholine produces at least a threefold increase in NO release. NO not only attenuated the baseline membrane oscillations but it also increased the magnitude of current required to generate the characteristic membrane potential oscillations. This resulted in a rightward shift in the frequency-current relationship and altered the excitability of hair cells. Our data suggest that these effects ensue because NO reduces whole cell Ca(2+) current and drastically decreases the open probability of single-channel events of the L-type and non L-type Ca(2+) channels in hair cells, an effect that is mediated through direct nitrosylation of the channel and activation of protein kinase G. Finally, NO increases the magnitude of Ca(2+)-activated K(+) currents via direct NO nitrosylation. We conclude that NO-mediated inhibition serves as a component of efferent nerve modulation of hair cells.