Eunyoung Yi

The origin of spontaneous activity in the developing auditory system.

Spontaneous activity in the developing auditory system is required for neuronal survival as well as the refinement and maintenance of tonotopic maps in the brain. However, the mechanisms responsible for initiating auditory nerve firing in the absence of sound have not been determined. Here we show that supporting cells in the developing rat cochlea spontaneously release ATP, which causes nearby inner hair cells to depolarize and release glutamate, triggering discrete bursts of action potentials in primary auditory neurons. This endogenous, ATP-mediated signalling synchronizes the output of neighbouring inner hair cells, which may help refine tonotopic maps in the brain. Spontaneous ATP-dependent signalling rapidly subsides after the onset of hearing, thereby preventing this experience-independent activity from interfering with accurate encoding of sound. These data indicate that supporting cells in the organ of Corti initiate electrical activity in auditory nerves before hearing, pointing to an essential role for peripheral, non-sensory cells in the development of central auditory pathways.

Sensorineural Deafness and Seizures in Mice Lacking Vesicular Glutamate Transporter 3

The expression of unconventional vesicular glutamate transporter VGLUT3 by neurons known to release a different classical transmitter has suggested novel roles for signaling by glutamate, but this distribution has raised questions about whether the protein actually contributes to glutamate release. We now report that mice lacking VGLUT3 are profoundly deaf due to the absence of glutamate release from hair cells at the first synapse in the auditory pathway. The early degeneration of some cochlear ganglion neurons in knockout mice also indicates an important developmental role for the glutamate released by hair cells before the onset of hearing. In addition, the mice exhibit primary, generalized epilepsy that is accompanied by remarkably little change in ongoing motor behavior. The glutamate release conferred by expression of VGLUT3 thus has an essential role in both function and development of the auditory pathway, as well as in the control of cortical excitability.

Two Modes of Release Shape the Postsynaptic Response at the Inner Hair Cell Ribbon Synapse

Cochlear inner hair cells (IHCs) convert sounds into receptor potentials and via their ribbon synapses into firing rates in auditory nerve fibers. Multivesicular release at individual IHC ribbon synapses activates AMPA-mediated EPSCs with widely ranging amplitudes. The underlying mechanisms and specific role for multivesicular release in encoding sound are not well understood. Here we characterize the waveforms of individual EPSCs recorded from afferent boutons contacting IHCs and compare their characteristics in immature rats (postnatal days 8–11) and hearing rats (postnatal days 19–21). Two types of EPSC waveforms were found in every recording: monophasic EPSCs, with sharp rising phases and monoexponential decays, and multiphasic EPSCs, exhibiting inflections on rising and decaying phases. Multiphasic EPSCs exhibited slower rise times and smaller amplitudes than monophasic EPSCs. Both types of EPSCs had comparable charge transfers, suggesting that they were activated by the release of similar numbers of vesicles, which for multiphasic EPSCs occurred in a less coordinated manner. On average, a higher proportion of larger, monophasic EPSCs was found in hearing compared to immature rats. In addition, EPSCs became significantly faster with age. The developmental increase in size and speed could improve auditory signaling acuity. Multiphasic EPSCs persisted in hearing animals, in some fibers constituting half of the EPSCs. The proportion of monophasic versus multiphasic EPSCs varied widely across fibers, resulting in marked heterogeneity of amplitude distributions. We propose that the relative contribution of two modes of multivesicular release, generating monophasic and multiphasic EPSCs, may underlie fundamental characteristics of auditory nerve fibers.

Differentiation of inner ear stem cells to functional sensory neurons.

Inner ear stem cells can be isolated by neurosphere formation from the vestibular organs and the cochlea. The cells are pluripotent, with the potential to become hair cells and neurons, the cochlear cell types whose loss causes deafness. Here we describe the control of cell fate decisions that determine the phenotype adopted by these progenitors, and we determine whether differentiation to sensory neurons is preferred over other types of neurons. Differentiation of progenitor cells recapitulated developmental pathways of embryonic sensory neurons. Based on marker expression, retinoic acid increased the yield of neurons and the percentage of sensory neurons obtained and caused a sharp increase in Pax2, a key transcription factor of cranial placodes. Markers of embryonic auditory and other sensory neurons, GATA3, Brn3a, and islet1, could be detected after 3 days of differentiation of the cells, and markers of the sensory phenotype, peripherin, calretinin, TrkC, and TrkB were expressed after 10 days. The differentiated cells had tetrodotoxin‐sensitive sodium currents and fired action potentials, and recordings revealed functional AMPA type‐glutamate receptors, further indicating that these cells had developed neuronal features. Neurons differentiated from these stem cells grew processes to hair cells in vitro. Development of functional activity in cells with the markers of sensory neurons suggested that the inner ear stem cells might have the capacity to replace cells lost due to neural degeneration.

Dendritic HCN Channels Shape Excitatory Postsynaptic Potentials at the Inner Hair Cell Afferent Synapse in the Mammalian Cochlea

Synaptic transmission at the inner hair cell (IHC) afferent synapse, the first synapse in the auditory pathway, is specialized for rapid and reliable signaling. Here we investigated the properties of a hyperpolarization-activated current (I(h)), expressed in the afferent dendrite of auditory nerve fibers, and its role in shaping postsynaptic activity. We used whole cell patch-clamp recordings from afferent dendrites directly where they contact the IHC in excised postnatal rat cochlear turns. Excitatory postsynaptic potentials (EPSPs) of variable amplitude (1-35 mV) were found with 10-90% rise times of about 1 ms and time constants of decay of about 5 ms at room temperature. Current-voltage relations recorded in afferent dendrites revealed I(h). The pharmacological profile and reversal potential (-45 mV) indicated that I(h) is mediated by hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels. The HCN channel subunits HCN1, HCN2, and HCN4 were found to be expressed in afferent dendrites using immunolabeling. Raising intracellular cAMP levels sped up the activation kinetics, increased the magnitude of I(h) and shifted the half activation voltage (V(half)) to more positive values (-104 +/- 3 to -91 +/- 2 mV). Blocking I(h) with 50 microM ZD7288 resulted in hyperpolarization of the resting membrane potential (approximately 4 mV) and slowing the decay of the EPSP by 47%, suggesting that I(h) is active at rest and shortens EPSPs, thereby potentially improving rapid and reliable signaling at this first synapse in the auditory pathway.

The Glutamate–Aspartate Transporter GLAST Mediates Glutamate Uptake at Inner Hair Cell Afferent Synapses in the Mammalian Cochlea

Ribbon synapses formed between inner hair cells (IHCs) and afferent dendrites in the mammalian cochlea can sustain high rates of release, placing strong demands on glutamate clearance mechanisms. To investigate the role of transporters in glutamate removal at these synapses, we made whole-cell recordings from IHCs, afferent dendrites, and glial cells adjacent to IHCs [inner phalangeal cells (IPCs)] in whole-mount preparations of rat organ of Corti. Focal application of the transporter substrate d-aspartate elicited inward currents in IPCs, which were larger in the presence of anions that permeate the transporter-associated anion channel and blocked by the transporter antagonist d,l-threo-β-benzyloxyaspartate. These currents were produced by glutamate–aspartate transporters (GLAST) (excitatory amino acid transporter 1) because they were weakly inhibited by dihydrokainate, an antagonist of glutamate transporter-1 (excitatory amino acid transporter 2) and were absent from IPCs in GLAST−/− cochleas. Furthermore, d-aspartate-induced currents in outside-out patches from IPCs exhibited larger steady-state currents than responses elicited by l-glutamate, a prominent feature of GLAST, and examination of cochlea from GLAST–Discosoma red (DsRed) promoter reporter mice revealed that DsRed expression was restricted to IPCs and other supporting cells surrounding IHCs. Saturation of transporters by photolysis of caged d-aspartate failed to elicit transporter currents in IHCs, as did local application of d-aspartate to afferent terminals, indicating that neither presynaptic nor postsynaptic membranes are major sites for glutamate removal. These data indicate that GLAST in supporting cells is responsible for transmitter uptake at IHC afferent synapses.

Autonomic pathways regulating pancreatic exocrine secretion

The parasympathetic (PNS) and sympathetic (SNS) and nervous systems densely innervate the exocrine pancreas. Efferent PNS pathways, consisting of central dorsal motor nucleus of the vagus (DMV) and peripheral pancreatic neurons, stimulate exocrine secretion. The DMV integrates cortical (olfactory, gustatory) and gastric, and intestinal vagal afferent input to determine central PNS outflow during cephalic, gastric and intestinal phases of exocrine secretion. Pancreatic neurons integrate DMV input with peripheral enteric, sympathetic, and, possibly, afferent axon reflexes to determine final PNS input to all exocrine effectors. Gut and islet hormones appear to modulate both central and peripheral PNS pathways. Preganglionic sympathetic neurons in the intermediolateral (IML) column of the spinal cord receive inputs from brain centers, some shared with the PNS, and innervate postganglionic neurons, mainly in prevertebral ganglia. Sympathetic innervation of the exocrine pancreas is primarily indirect, and inhibits secretion by decreasing blood flow and inhibiting transmission in pancreatic ganglia. Interactions between SNS and PNS pathways appear to occur in brain, spinal cord, pancreatic and prevertebral ganglia, and at neuroeffector synapses. Thus, the PNS and SNS pathways regulating the exocrine pancreas are directly or indirectly antagonistic at multiple sites: the state of exocrine secretion reflects the balance of these influences. Despite over a century of study, much remains to be understood about the connections of specific neurons forming pancreatic pathways, their processes of neurotransmission, and how disruption of these pathways contributes to pancreatic disease.

Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the postsynaptic activity of a single ribbon synapse 1-4. Ribbon synapses of sensory cells release neurotransmitter continuously, the rate of which is modulated in response to graded changes in IHC membrane potential 5. Ribbon synapses have been shown to operate by multivesicular release, where multiple vesicles can be released simultaneously to evoke excitatory postsynaptic currents (EPSCs) of varying amplitudes 1, 4, 6-11. Neither the role of the presynaptic ribbon, nor the mechanism underlying multivesicular release is currently well understood. The IHC is innervated by 10-20 auditory nerve fibers, and every fiber contacts the IHC with a unmyelinated single ending to form a single ribbon synapse. The small size of the afferent boutons contacting IHCs (approximately 1 μm in diameter) enables recordings with exceptional temporal resolution to be made. Furthermore, the technique can be adapted to record from both pre- and postsynaptic cells simultaneously, allowing the transfer function at the synapse to be studied directly 2. This method therefore provides a means by which fundamental aspects of neurotransmission can be studied, from multivesicular release to the elusive function of the ribbon in sensory cells.

Catecholamines and 5-hydroxytryptamine in tissues of the rabbit exocrine pancreas.

Objectives: Norepinephrine (NE), dopamine (DA), epinephrine (Epi), and 5-hydroxytryptamine (5-HT) all modulate pancreatic exocrine secretion, yet their concentrations in specific tissues of the exocrine pancreas are unknown. Methods: Concentrations of catecholamines and 5-HT in rabbit pancreatic ganglia, acini, ducts and ampullae, and arteries and veins were measured using HPLC. Results: Concentrations of NE in ganglia from the head/neck region were significantly higher than those from the body (1620 ± 220 vs. 778 ± 179 pmol/mg protein). Acini contained little NE, DA, or 5-HT (9 ± 2, 0.9 ± 0.2, 13 ± 5 pmol/mg protein). Ducts and ampullae contained NE (314 ± 74 and 156 ± 24 pmol/mg protein), DA (43 ± 14 and 13 ± 4 pmol/mg protein), Epi (63 ± 29 and 39 ± 6 pmol/mg protein), and 5-HT (696 ± 151 and 3563 ± 288 pmol/mg protein). Arteries and veins contained the highest concentrations of NE (1962 ± 463 and 736 ± 80 pmol/mg protein, respectively). Conclusions: Pancreatic ganglia and blood vessels, rather than acini, are the main sites of noradrenergic sympathetic innervation of the rabbit exocrine pancreas. These nerves preferentially target ganglionic transmission in the head/neck versus the body. Serotonergic nerves provide little or no innervation of rabbit pancreatic ganglia or acini.