RESUMEN
The hair bundle is the mechanosensory organelle of hair cells that detects mechanical stimuli caused by sounds, head motions, and fluid flows. Each hair bundle is an assembly of cellular-protrusions called stereocilia, which differ in height to form a staircase. Stereocilia have different heights, widths, and separations in different species, sensory organs, positions within an organ, hair-cell types, and even within a single hair bundle. The dimensions of the stereociliary assembly dictate how the hair bundle responds to stimuli. These hair-bundle properties have been measured previously only to a limited degree. In particular, mammalian data are either incomplete, lack control for age or position within an organ, or have artifacts owing to fixation or dehydration. Here, we provide a complete set of measurements for postnatal day (P) 11 C57BL/6J mouse apical inner hair cells (IHCs) obtained from living tissue, tissue mildly-fixed for fluorescent imaging, or tissue strongly fixed and dehydrated for scanning electronic microscopy (SEM). We found that hair bundles mildly-fixed for fluorescence had the same dimensions as living hair bundles, whereas SEM-prepared hair bundles shrank uniformly in stereociliary heights, widths, and separations. By determining the shrinkage factors, we imputed live dimensions from SEM that were too small to observe optically. Accordingly, we created the first complete blueprint of a living IHC hair bundle. We show that SEM-prepared measurements strongly affect calculations of a bundle's mechanical properties - overestimating stereociliary deflection stiffness and underestimating the fluid coupling between stereocilia. The methods of measurement, the data, and the consequences we describe illustrate the high levels of accuracy and precision required to understand hair-bundle mechanotransduction.
RESUMEN
Estrogens support major brain functions including cognition, reproduction, neuroprotection and sensory processing. Neuroestrogens are synthesized within some brain areas by the enzyme aromatase and can rapidly modulate local circuit functions, yet the cellular physiology and sensory-response profiles of aromatase neurons are essentially unknown. In songbirds, social and acoustic stimuli drive neuroestrogen elevations in the auditory forebrain caudomedial nidopallium (NCM). In both males and females, neuroestrogens rapidly enhance NCM auditory processing and auditory learning. Estrogen-producing neurons in NCM may therefore exhibit distinguishing profiles for sensory-activation and intrinsic electrophysiology. Here, we explored these questions using both immunocyctochemistry and electrophysiological recordings. Immunoreactivity for aromatase and the immediate early gene EGR1, a marker of activity and plasticity, were quantified in NCM of song-exposed animals versus silence-exposed controls. Using whole-cell patch clamp recordings from NCM slices, we also documented the intrinsic excitability profiles of aromatase-positive and aromatase-negative neurons. We observed that a subset of aromatase neurons were significantly activated during song playback, in both males and females, and in both hemispheres. A comparable population of non-aromatase-expressing neurons were also similarly driven by song stimulation. Membrane properties (i.e., resting membrane potential, rheobase, input resistance and multiple action potential parameters) were similarly indistinguishable between NCM aromatase and non-aromatase neurons. Together, these findings demonstrate that aromatase and non-aromatase neurons in NCM are indistinct in terms of their intrinsic electrophysiology and responses to song. Nevertheless, such similarities in response properties may belie more subtle differences in underlying conductances and/or computational roles that may be crucial to their function.
