Electrophysiological properties of neurons grown on soft polymer scaffolds reveal the potential to develop neuromimetic culture environments.

Tissue engineering methodologies for various physiological systems are seeing a significant trend towards 3D cell culture in or on 'soft' polymeric hydrogel materials, widely considered to provide a more biomimetic environment for cell growth versus 'hard' materials such as glass or plastic. Progress has been slower with 3D neural cell culture with current studies overwhelmingly reliant on hard substrates. Accordingly, our knowledge of the alterations in electrochemical properties of neurons propagated in soft materials is relatively limited. In this study, primary cortical neurons and glial cells were seeded onto the surface of collagen hydrogels and grown in vitro for 7-8 days. At this time, neurons had formed a complex neurite web interspersed with astrocytes. Neuronal patch clamp recordings revealed voltage-gated Na+ and K+ currents in voltage clamp and action potentials in current clamp. When measured at voltages close to maximum activation, both currents were >1 nA in mean amplitude. When compared to primary cortical neurons cultured on glass coverslips, but otherwise under similar conditions (Evans et al., 2017), the Na+ current from hydrogel neurons was found to be significantly larger although there were no differences in the K+ current amplitude, membrane potential, input resistance or cell capacitance. We speculate that the larger size of the neuronal voltage-dependent Na+ current in the hydrogels is related to the better biomimetic properties of the soft material, being close to values reported for neurons recorded in brain slices. The results highlight the potential benefits offered by neuronal culture on soft and biomimetic polymeric materials for neural tissue engineering studies.

Conductance properties of the acetylcholine receptor current of Guinea pig outer hair cells.

The nicotinic acetylcholine receptor (AChR) current of outer hair cells (OHCs) was investigated in isolated and voltage-clamped cells under conditions where co-activating Ca(2+)-activated K(+) currents had been abolished using internal BAPTA, external calcium removal and/or depolarisation to positive voltages. The AChR current activated rapidly and thereafter declined in the continued presence of ACh. Reversal potential measurements indicated that it was a non-specific cation current with a substantial Ca(2+) permeability. It had a characteristic bidirectional rectification with an especially prominent outward component in solutions containing 1 mM Ca(2+). The I-V relation was fitted with a single-energy barrier model. The fit suggests a blocking site within the channel, situated about one third of the way through the membrane from the outside and probably normally occupied by Ca(2+) or Mg(2+). The AChR current was sensitive to the external Ca(2+) since it was reduced, to differing extents, in nominally Ca(2+)-free saline or in high Ca(2+) saline (10 mM). In the presence of a nominally Mg(2+)-free solution containing 0.4 mM Ca(2+), the currents were larger, indicating a potentiated response. This type of behaviour is also shown by recombinant α9α10 AChRs, suggesting a close similarity. The AChR current at both positive and negative voltages was reduced in external solutions where most of the Na(+) had been replaced by NMG(+). The conductance properties of the OHC AChR are compared with α9α10 receptors and nicotinic receptors in other hair cells and discussed in terms of the accepted functional role of providing calcium influx leading to efferent synaptic inhibition of hair cells.

Localisation of the mechanotransducer channels in mammalian cochlear hair cells provides clues to their gating.

Our sense of hearing and balance relies on the very rapid gating of mechanotransducer channels known to be located close to the tops of the hair cell stereocilia within the stereociliary bundle. The molecular identity of the channels is unknown but functional aspects such as permeation, block and sensitivity to bundle displacement are well known. The channel has high calcium permeability and this feature has been used in conjunction with fast confocal calcium imaging to unambiguously localise the channels at the top of the two shorter rows of stereocilia in mammalian cochlear hair cells. The data suggest that they are completely absent from the tallest row. It is thought that the structures connecting stereocilia in adjacent rows, the tip links, are either directly responsible for the channel's mechanical gating, or are closely associated with the gating process. The channels must therefore be associated with the bottom part of the tip links and not the top. This feature has important implications for both the channel's gating mechanism and its regulatory adaptation mechanism. The tip link remains an attractive candidate for mechanical coupling between the bundle and the channel or an accessory protein. The localisation of the mechanotransducer channels to the lower end of the tip link represents an important milestone in the journey towards eventual identification of the channel and its gating mechanism.

A large-conductance calcium-selective mechanotransducer channel in mammalian cochlear hair cells.

Sound stimuli are detected in the cochlea by opening of hair cell mechanotransducer (MT) channels, one of the few ion channels not yet conclusively identified at a molecular level. To define their performance in situ, we measured MT channel properties in inner hair cells (IHCs) and outer hair cells (OHCs) at two locations in the rat cochlea tuned to different characteristic frequencies (CFs). The conductance (in 0.02 mM calcium) of MT channels from IHCs was estimated as 260 pS at both low-frequency and mid-frequency positions, whereas that from OHCs increased with CFs from 145 to 210 pS. The combination of MT channel conductance and tip link number, assayed from scanning electron micrographs, accounts for variation in whole-cell current amplitude for OHCs and its invariance for IHCs. Channels from apical IHCs and OHCs having a twofold difference in unitary conductance were both highly calcium selective but were distinguishable by a small but significant difference in calcium permeability and in their response to lowering ionic strength. The results imply that the MT channel has properties possessed by few known candidates, and its diversity suggests expression of multiple isoforms.

Depolarization of cochlear outer hair cells evokes active hair bundle motion by two mechanisms.

There is current debate about the origin of mechanical amplification whereby outer hair cells generate force to augment the sensitivity and frequency selectivity of the mammalian cochlea. To distinguish contributions to force production from the mechanotransducer (MET) channels and somatic motility, we have measured hair bundle motion during depolarization of individual outer hair cells in isolated rat cochleas. Depolarization evoked rapid positive bundle deflections that were reduced by perfusion with the MET channel blocker dihydrostreptomycin, with no effect on the nonlinear capacitance that is a manifestation of prestin-driven somatic motility. However, the movements were also diminished by Na salicylate and depended on the intracellular anion, properties implying involvement of the prestin motor. Furthermore, depolarization of one outer hair cell caused motion of neighboring hair bundles, indicating overall motion of the reticular lamina. Depolarization of solitary outer hair cells caused cell-length changes whose voltage-activation range depended on the intracellular anion but were insensitive to dihydrostreptomycin. These results imply that both the MET channels and the somatic motor participate in hair bundle motion evoked by depolarization. It is conceivable that the two processes can interact, a signal from the MET channels being capable of modulating the activity of the prestin motor.

Fast adaptation of mechanoelectrical transducer channels in mammalian cochlear hair cells.

Outer hair cells are centrally involved in the amplification and frequency tuning of the mammalian cochlea, but evidence about their transducing properties in animals with fully developed hearing is lacking. Here we describe measurements of mechanoelectrical transducer currents in outer hair cells of rats between postnatal days 5 and 18, before and after the onset of hearing. Deflection of hair bundles using a new rapid piezoelectric stimulator evoked transducer currents with ultra-fast activation and adaptation kinetics. Fast adaptation resembled the same process in turtle hair cells, where it is regulated by changes in stereociliary calcium. It is argued that sub-millisecond transducer adaptation can operate in outer hair cells under the ionic, driving force and temperature conditions that prevail in the intact mammalian cochlea.