Anthony J. Ricci

Localization of inner hair cell mechanotransducer channels using high speed calcium imaging

Hair cells detect vibrations of their stereociliary bundle by activation of mechanically sensitive transducer channels. Although evidence suggests the transducer channels are near the stereociliary tops and are opened by force imparted by tip links connecting contiguous stereocilia, the exact channel site remains controversial. We used fast confocal imaging of fluorescence changes reflecting calcium entry during bundle stimulation to localize the channels. Calcium signals were visible in single stereocilia of rat cochlear inner hair cells and were up to tenfold larger and faster in the second and third stereociliary rows than in the tallest first row. The number of functional stereocilia was proportional to transducer current amplitude, indicating that there were about two channels per stereocilium. Comparable results were obtained in outer hair cells. The observations, supported by theoretical simulations, suggest there are no functional mechanically sensitive transducer channels in first row stereocilia and imply the channels are present only at the bottom of the tip links.

Mechanosensitive Hair Cell-like Cells from Embryonic and Induced Pluripotent Stem Cells

Mechanosensitive sensory hair cells are the linchpin of our senses of hearing and balance. The inability of the mammalian inner ear to regenerate lost hair cells is the major reason for the permanence of hearing loss and certain balance disorders. Here, we present a stepwise guidance protocol starting with mouse embryonic stem and induced pluripotent stem cells, which were directed toward becoming ectoderm capable of responding to otic-inducing growth factors. The resulting otic progenitor cells were subjected to varying differentiation conditions, one of which promoted the organization of the cells into epithelial clusters displaying hair cell-like cells with stereociliary bundles. Bundle-bearing cells in these clusters responded to mechanical stimulation with currents that were reminiscent of immature hair cell transduction currents.

Functional auditory hair cells produced in the mammalian cochlea by in utero gene transfer

Sensory hair cells in the mammalian cochlea convert mechanical stimuli into electrical impulses that subserve audition. Loss of hair cells and their innervating neurons is the most frequent cause of hearing impairment. Atonal homologue 1 (encoded by Atoh1, also known as Math1) is a basic helix–loop–helix transcription factor required for hair-cell development, and its misexpression in vitro and in vivo generates hair-cell-like cells. Atoh1-based gene therapy to ameliorate auditory and vestibular dysfunction has been proposed. However, the biophysical properties of putative hair cells induced by Atoh1 misexpression have not been characterized. Here we show that in utero gene transfer of Atoh1 produces functional supernumerary hair cells in the mouse cochlea. The induced hair cells display stereociliary bundles, attract neuronal processes and express the ribbon synapse marker carboxy-terminal binding protein 2 (refs 12,13). Moreover, the hair cells are capable of mechanoelectrical transduction and show basolateral conductances with age-appropriate specializations. Our results demonstrate that manipulation of cell fate by transcription factor misexpression produces functional sensory cells in the postnatal mammalian cochlea. We expect that our in utero gene transfer paradigm will enable the design and validation of gene therapies to ameliorate hearing loss in mouse models of human deafness.

Role for gamma-aminobutyric acid in selective vulnerability in gerbils.

We tested the efficacy of various putative neuroprotective agents in the gerbil model of delayed neuronal death. The selective loss of anterior CA1 neurons of the hippocampus 4 days after 5 minutes of bilateral ischemia was complete in greater than 90% of the gerbils examined. We tested 11 agents for their ability to protect against neuronal loss. Only those agents that were associated with the GABAergic system exhibited protection and only when administered before the ischemic insult. The possibility that delayed neuronal death is the result of a primary defect in inhibitory neurotransmission is considered.

Tonotopic variation in the conductance of the hair cell mechanotransducer channel.

Hair cells in the vertebrate cochlea are arranged tonotopically with their characteristic frequency (CF), the sound frequency to which they are most sensitive, changing systematically with position. Single mechanotransducer channels of hair cells were characterized at different locations in the turtle cochlea. In 2.8 mM external Ca2+, the channels chord conductance was 118 pS (range 80-163 pS), which nearly doubled (range 149-300 pS) on reducing Ca2+ to 50 microM. In both Ca2+ concentrations, the conductance was positively correlated with hair cell CF. Variation in channel conductance can largely explain the increases in size of the macroscopic transducer current and speed of adaptation with CF. It suggests diversity of transducer channel structure or environment along the cochlea that may be an important element of its tonotopic organization.

Homeostatic Scaling of Vesicular Glutamate and GABA Transporter Expression in Rat Neocortical Circuits

Homeostatic control of pyramidal neuron firing rate involves a functional balance of feedforward excitation and feedback inhibition in neocortical circuits. Here, we reveal a dynamic scaling in vesicular excitatory (vesicular glutamate transporters VGLUT1 and VGLUT2) and inhibitory (vesicular inhibitory amino acid transporter VIAAT) transporter mRNA and synaptic protein expression in rat neocortical neuronal cultures, using a well established in vitro protocol to induce homeostatic plasticity. During the second and third week of synaptic differentiation, the predominant vesicular transporters expressed in neocortical neurons, VGLUT1 and VIAAT, are both dramatically upregulated. In mature cultures, VGLUT1 and VIAAT exhibit bidirectional and opposite regulation by prolonged activity changes. Endogenous coregulation during development and homeostatic scaling of the expression of the transporters in functionally differentiated cultures may serve to control vesicular glutamate and GABA filling and adjust functional presynaptic excitatory/inhibitory balance. Unexpectedly, hyperexcitation in differentiated cultures triggers a striking increase in VGLUT2 mRNA and synaptic protein, whereas decreased excitation reduces levels. VGLUT2 mRNA and protein are expressed in subsets of VGLUT1-encoded neocortical neurons that we identify in primary cultures and in neocortex in situ and in vivo. After prolonged hyperexcitation, downregulation of VGLUT1/synaptophysin intensity ratios at most synapses is observed, whereas a subset of VGLUT1-containing boutons selectively increase the expression of VGLUT2. Bidirectional and opposite regulation of VGLUT1 and VGLUT2 by activity may serve as positive or negative feedback regulators for cortical synaptic transmission. Intracortical VGLUT1/VGLUT2 coexpressing neurons have the capacity to independently modulate the level of expression of either transporter at discrete synapses and therefore may serve as a plastic interface between subcortical thalamic input (VGLUT2) and cortical output (VGLUT1) neurons.

Calcium permeation of the turtle hair cell mechanotransducer channel and its relation to the composition of endolymph

1 Recordings of mechanoelectrical transducer currents were combined with calcium imaging of hair bundles in turtle auditory hair cells located near the high‐frequency end of the cochlea. The external face of the hair bundles was perfused with a range of Ca2+ concentrations to study the quantitative relationship between Ca2+ influx and transducer adaptation. 2 With Na+ as the monovalent ion, the peak amplitude of the transducer current decreased monotonically as the external [Ca2+] was raised from 25 μM to 20 mM. When Na+ was replaced with the impermeant Tris the transducer current increased with external [Ca2+]. These results indicate that Ca2+ can both permeate and block the transducer channels. The Ca2+ concentration for half‐block of the monovalent current was 1 mM. 3 To quantify the Ca2+ influx, the fraction of transducer current carried by Ca2+ was measured using the change in bundle fluorescence in cells loaded with 1 mM Calcium Green‐1. The fluorescence change was calibrated by substituting an impermeable monovalent ion to render Ca2+ the sole charge carrier. 4 In the presence of Na+, the fractional Ca2+ current was ≈10 % in 50 μM Ca2+, a concentration similar to that in endolymph, which bathes the hair bundles in vivo. The amount of Ca2+ entering was dependent on the identity of the monovalent ion, and was larger with K+, suggesting that the transducer channel is a multi‐ion pore. 5 Over a range of ionic conditions, the rate of transducer adaptation was proportional to Ca2+ influx indicating that adaptation is driven by a rise in intracellular [Ca2+]. 6 Shifts in the current‐displacement function along the displacement axis in different external Ca2+ concentrations were predictable from variation in the resting Ca2+ influx. We suggest that changes in the resting open probability of the transducer channels adjust the entry of Ca2+ to keep its concentration constant at an internal site. 7 The results demonstrate that endolymph containing high K+, 50 μM Ca2+ and low Mg2+ concentrations, maximizes the transducer current while still allowing sufficient Ca2+ entry to drive adaptation. The hair cell mechanotransducer channel, in its permeation and block by Ca2+, shows behaviour similar to the voltage‐gated Ca2+ channel and the cyclic nucleotide‐gated channel.

Mechanisms of Aminoglycoside Ototoxicity and Targets of Hair Cell Protection

Aminoglycosides are commonly prescribed antibiotics with deleterious side effects to the inner ear. Due to their popular application as a result of their potent antimicrobial activities, many efforts have been undertaken to prevent aminoglycoside ototoxicity. Over the years, understanding of the antimicrobial as well as ototoxic mechanisms of aminoglycosides has increased. These mechanisms are reviewed in regard to established and potential future targets of hair cell protection.

The effects of calcium buffering and cyclic AMP on mechano-electrical transduction in turtle auditory hair cells

1 The effects of intracellular Ca2+ buffering on hair cell mechanotransduction were studied in an intact cochlear epithelium where the endolymphatic and perilymphatic surfaces could be separately perfused with different Ca2+ solutions. 2 The speed and extent of transducer adaptation increased as the concentration in the patch electrode of the Ca2+ buffer BAPTA was lowered. In 0.1 mm BAPTA or less, the transducer adapted almost completely, with a mean time constant of 0.8 ms. 3 For a fixed internal BAPTA concentration, the transducer conductance varied with hair cell location, increasing towards the high‐frequency end of the cochlea, and the time constant of adaptation decreased proportionally. At a given cochlear location, hair cells with larger transducer conductances displayed faster adaptation. We suggest that transducer adaptation accounts for a variable high‐pass filter observed in the acoustic tuning curve. 4 The effects of perfusion of 50 μm Ca2+ endolymph depended on the BAPTA concentration of the electrode: with 3 mm BAPTA, adaptation was abolished, but in most recordings with 0.01 or 0.1 mm BAPTA, rapid adaptation was retained. The current–displacement curve was also shifted less the lower the intracellular BAPTA concentration. Cells in the high‐frequency half of the papilla retained adaptation at a higher BAPTA concentration. 5 Treatment with the cAMP agonist, 8‐bromo‐cAMP, or with the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine, caused a rightward shift in the current–displacement curve which was independent of the internal BAPTA concentration. 6 We conclude that the free Ca2+ and cyclic nucleotide concentrations of the hair bundle modulate the position of the activation curve of the transducer. The factors which may be important for the correct functioning of adaptation in vivo are discussed.

Probing the pore of the auditory hair cell mechanotransducer channel in turtle

Hair cell mechano‐electric transducer (MET) channels play a pivotal role in auditory and vestibular signal detection, yet few data exist regarding their molecular nature. Present work characterizes the MET channel pore, a region whose properties are thought to be intrinsically determined. Two approaches were used. First, the channel was probed with antagonists of candidate channel subtypes including: cyclic nucleotide‐gated channels, transient receptor potential channels and gap‐junctional channels. Eight new antagonists were identified. Most of the effective antagonists had a partially charged amine group predicted to penetrate the channel pore, antagonizing current flow, while the remainder of the molecule prevented further permeation of the compound through the pore. This blocking mechanism was tested using curare to demonstrate the open channel nature of the block and by identifying methylene blue as a permeant channel blocker. The second approach estimated dimensions of the channel pore with simple amine compounds. The narrowest diameter of the pore was calculated as 12.5 ± 0.8  Å and the location of a binding site ∼45% of the way through the membrane electric field was calculated. Channel length was estimated as ∼31 Å and the width of the pore mouth at < 17  Å. Each effective antagonist had a minimal diameter, measured about the penetrating amine, of less than the pore diameter, with a direct correlation between IC50 and minimal diameter. The IC50 was also directly related to the length of the amine side chains, further validating the proposed pore blocking mechanism. Data provided by these two approaches support a hypothesis regarding channel permeation and block that incorporates molecular dimensions and ion interactions within the pore.