Inner ear biomechanics reveals a Late Triassic origin for mammalian endothermy.

Endothermy underpins the ecological dominance of mammals and birds in diverse environmental settings1,2. However, it is unclear when this crucial feature emerged during mammalian evolutionary history, as most of the fossil evidence is ambiguous3-17. Here we show that this key evolutionary transition can be investigated using the morphology of the endolymph-filled semicircular ducts of the inner ear, which monitor head rotations and are essential for motor coordination, navigation and spatial awareness18-22. Increased body temperatures during the ectotherm-endotherm transition of mammal ancestors would decrease endolymph viscosity, negatively affecting semicircular duct biomechanics23,24, while simultaneously increasing behavioural activity25,26 probably required improved performance27. Morphological changes to the membranous ducts and enclosing bony canals would have been necessary to maintain optimal functionality during this transition. To track these morphofunctional changes in 56 extinct synapsid species, we developed the thermo-motility index, a proxy based on bony canal morphology. The results suggest that endothermy evolved abruptly during the Late Triassic period in Mammaliamorpha, correlated with a sharp increase in body temperature (5-9 °C) and an expansion of aerobic and anaerobic capacities. Contrary to previous suggestions3-14, all stem mammaliamorphs were most probably ectotherms. Endothermy, as a crucial physiological characteristic, joins other distinctive mammalian features that arose during this period of climatic instability28.

Novel cell types and developmental lineages revealed by single-cell RNA-seq analysis of the mouse crista ampullaris.

This study provides transcriptomic characterization of the cells of the crista ampullaris, sensory structures at the base of the semicircular canals that are critical for vestibular function. We performed single-cell RNA-seq on ampullae microdissected from E16, E18, P3, and P7 mice. Cluster analysis identified the hair cells, support cells and glia of the crista as well as dark cells and other nonsensory epithelial cells of the ampulla, mesenchymal cells, vascular cells, macrophages, and melanocytes. Cluster-specific expression of genes predicted their spatially restricted domains of gene expression in the crista and ampulla. Analysis of cellular proportions across developmental time showed dynamics in cellular composition. The new cell types revealed by single-cell RNA-seq could be important for understanding crista function and the markers identified in this study will enable the examination of their dynamics during development and disease.

[Ultrastructural study of ampulla of mouse inner ear].

Objective: To observe the ultrastructure of the ampulla, and analyze its physiological and pathological significance. Methods: In this study, 20 Kunming mice were used, and scanning electron microscopy was used to observe the ultrastructure of the ampulla of inner ear. Results: Otoconia was found among the cilia bundles of different haircell(intercilla otoconia of ampulla). The cupula was attached to the lateral wall of the ampulla, and easily to be separated; after separated, a kind of slender crystal(surface otoconia of ampulla) could be seen between the cupula and lateral wall of the ampulla, both sides of ampullary crest were covered with slender crystals too. On the canal side of the ampulla wall, there was more particulate matter attached to the wall near the bottom of ampullary crest, partially embedded in the wall, and less on the utricle side of the ampulla wall. Conclusions: The observation of the ultrastructure of the ampulla is helpful for better understanding the physiological functions of the semicircular canals and the ampulla, and better understanding the pathogenesis and solution of some vertigo diseases.

Morphological basis for a tonotopic design of an insect ear.

The tonotopically organized hearing organs of bushcrickets provide the opportunity for a detailed correlation of morphological and structural properties within hearing organs that are needed to establish tonotopic gradients. In the present study of a tonotopic insect hearing organ, we combine mechanical measurements of sound-induced hearing organ motion and detailed anatomical investigations to explore the anatomical basis of tonotopy. We compare mechanical data of frequency responses along the auditory organ to several anatomical parameters. Low frequency responses are related to larger organ and cap cell size in the proximal part of the hearing organ while in the distal part of the organ, small organ and cap cell size is related to high-frequency representation. However, the correlation between organ and cap cell size with continuous frequency representation along the organ is not very tight. Instead, the height of the organ and the corresponding length of the sensory dendrites are best correlated to tonotopic frequency representation. The sensory dendrite contains a ciliary root with a pronounced cross-banding of electron-dense material that should be important for the stiffness of the dendrite. The geometry of surrounding structures like the hemolymph channel and the acoustic trachea as well as the extension of the tectorial membrane are not correlated to the tonotopy. We provide evidence that tonotopy in the bushcricket hearing organ may depend on the size of ciliary structures. In particular, the ciliary root of the sensory cells is a likely cellular basis of tonotopy.

Assessing morphology and function of the semicircular duct system: introducing new in-situ visualization and software toolbox.

The semicircular duct system is part of the sensory organ of balance and essential for navigation and spatial awareness in vertebrates. Its function in detecting head rotations has been modelled with increasing sophistication, but the biomechanics of actual semicircular duct systems has rarely been analyzed, foremost because the fragile membranous structures in the inner ear are hard to visualize undistorted and in full. Here we present a new, easy-to-apply and non-invasive method for three-dimensional in-situ visualization and quantification of the semicircular duct system, using X-ray micro tomography and tissue staining with phosphotungstic acid. Moreover, we introduce Ariadne, a software toolbox which provides comprehensive and improved morphological and functional analysis of any visualized duct system. We demonstrate the potential of these methods by presenting results for the duct system of humans, the squirrel monkey and the rhesus macaque, making comparisons with past results from neurophysiological, oculometric and biomechanical studies. Ariadne is freely available at http://www.earbank.org.

Crista egregia: a geometrical model of the crista ampullaris, a sensory surface that detects head rotations.

The crista ampullaris is the epithelium at the end of the semicircular canals in the inner ear of vertebrates, which contains the sensory cells involved in the transduction of the rotational head movements into neuronal activity. The crista surface has the form of a saddle, or a pair of saddles separated by a crux, depending on the species and the canal considered. In birds, it was described as a catenoid by Landolt et al. (J Comp Neurol 159(2):257-287, doi: 10.1002/cne.901590207 , 1972). In the present work, we establish that this particular form results from principles of invariance maximization and energy minimization. The formulation of the invariance principle was inspired by Takumida (Biol Sci Space 15(4):356-358, 2001). More precisely, we suppose that in functional conditions, the equations of linear elasticity are valid, and we assume that in a certain domain of the cupula, in proximity of the crista surface, (1) the stress tensor of the deformed cupula is invariant under the gradient of the pressure, (2) the dissipation of energy is minimum. Then, we deduce that in this domain the crista surface is a minimal surface and that it must be either a planar, or helicoidal Scherk surface, or a piece of catenoid, which is the unique minimal surface of revolution. If we add the hypothesis that the direction of invariance of the stress tensor is unique and that a bilateral symmetry of the crista exists, only the catenoid subsists. This finding has important consequences for further functional modeling of the role of the vestibular system in head motion detection and spatial orientation.

Anatomy of the lamprey ear: morphological evidence for occurrence of horizontal semicircular ducts in the labyrinth of Petromyzon marinus.

In jawed (gnathostome) vertebrates, the inner ears have three semicircular canals arranged orthogonally in the three Cartesian planes: one horizontal (lateral) and two vertical canals. They function as detectors for angular acceleration in their respective planes. Living jawless craniates, cyclostomes (hagfish and lamprey) and their fossil records seemingly lack a lateral horizontal canal. The jawless vertebrate hagfish inner ear is described as a torus or doughnut, having one vertical canal, and the jawless vertebrate lamprey having two. These observations on the anatomy of the cyclostome (jawless vertebrate) inner ear have been unchallenged for over a century, and the question of how these jawless vertebrates perceive angular acceleration in the yaw (horizontal) planes has remained open. To provide an answer to this open question we reevaluated the anatomy of the inner ear in the lamprey, using stereoscopic dissection and scanning electron microscopy. The present study reveals a novel observation: the lamprey has two horizontal semicircular ducts in each labyrinth. Furthermore, the horizontal ducts in the lamprey, in contrast to those of jawed vertebrates, are located on the medial surface in the labyrinth rather than on the lateral surface. Our data on the lamprey horizontal duct suggest that the appearance of the horizontal canal characteristic of gnathostomes (lateral) and lampreys (medial) are mutually exclusive and indicate a parallel evolution of both systems, one in cyclostomes and one in gnathostome ancestors.

[The effect of otolith entrances upon horizontal nystagmus induced by stimulation of semicircular ducts and retina in pigeons].

The semicircular ducts in pigeons were stimulated either in an isolated way (the angular acceleration being 10 degrees/c2) or together with the otolith organs (the peak value of the interaural acceleration being 0.5 g). In one and the same situation, both the inhibitory and the activating otolith effects on nystagmus were found. In one and the same animal, changes of the postoptokinetic and canal nystagmuses could be qualitatively different. The results obtained contradict the hypothesis of changes in the "velocity accumulator" time constants as the only cause of changes in the postoptokinetic and rotatory nystagmuses under conditions of low-frequency otolith stimulation.

Infrared photostimulation of the crista ampullaris.

The present results show that the semicircular canal crista ampullaris of the toadfish, Opsanus tau, is sensitive to infrared radiation (IR) applied in vivo. IR pulse trains (∼1862 nm, ∼200 µs pulse⁻¹) delivered to the sensory epithelium by an optical fibre evoked profound changes in phasic and tonic discharge rates of postsynaptic afferent neurons. Phasic afferent responses to pulsed IR occurred with a latency of <8 ms while tonic responses developed with a time constant (τ) of 7 ms to 10 s following the onset or cessation of the radiation. Afferents responded to direct optical radiation of the sensory epithelium but did not respond to thermal stimuli that generated nearly equivalent temperature increases of the whole organ. A subset of afferent neurons fired an action potential in response to each IR pulse delivered to the sensory epithelium, at phase-locked rates up to 96 pulses per second. The latency between IR pulses and afferent nerve action potentials was much greater than synaptic delay and spike generation, demonstrating the presence of a signalling delay interposed between the IR pulse and the action potential. The same IR stimulus applied to afferent nerve axons failed to evoke responses of similar magnitude and failed to phase-lock afferent nerve action potentials. The present data support the hypothesis that pulsed IR activates sensory hair cells, thus leading to modulation of synaptic transmission and afferent nerve discharge reported here.

Dynamics of glutamatergic synapses in the medial vestibular nucleus of the mouse.

During sinusoidal rotation or translation, primary vestibular afferents modulate their discharge rates at the frequency of motion, effectively transmitting frequency-modulated (FM) signals. This study indicates a possible role for excitatory synapses in the processing of FM signals by vestibular brainstem pathways. Inputs to medial vestibular neurons were activated with FM pulse trains, while inhibitory transmission was blocked. The relationship between the presynaptic pulse rate and the postsynaptic membrane potential was found to be linear within a range of pulse rates. Short-term plasticity was a factor contributing to sensitivity at higher modulating frequencies. The amount of low-pass filtering was correlated with excitatory postsynaptic potential (EPSP) shape, which affected temporal summation during the train. Although the NMDA component of glutamatergic transmission affected EPSP shape, it made only a minor contribution to the dynamics of synaptic transmission. Most responses showed low-pass filtering over the entire 1-16 Hz range. Overall, excitatory synapses in the medial vestibular nucleus contribute a low-pass filter to central vestibular processing and complement the high-pass filtering that is introduced both by peripheral vestibular dynamics and by the intrinsic dynamics of secondary vestibular neurons.

Bmp4 is essential for the formation of the vestibular apparatus that detects angular head movements.

Angular head movements in vertebrates are detected by the three semicircular canals of the inner ear and their associated sensory tissues, the cristae. Bone morphogenetic protein 4 (Bmp4), a member of the Transforming growth factor family (TGF-beta), is conservatively expressed in the developing cristae in several species, including zebrafish, frog, chicken, and mouse. Using mouse models in which Bmp4 is conditionally deleted within the inner ear, as well as chicken models in which Bmp signaling is knocked down specifically in the cristae, we show that Bmp4 is essential for the formation of all three cristae and their associated canals. Our results indicate that Bmp4 does not mediate the formation of sensory hair and supporting cells within the cristae by directly regulating genes required for prosensory development in the inner ear such as Serrate1 (Jagged1 in mouse), Fgf10, and Sox2. Instead, Bmp4 most likely mediates crista formation by regulating Lmo4 and Msx1 in the sensory region and Gata3, p75Ngfr, and Lmo4 in the non-sensory region of the crista, the septum cruciatum. In the canals, Bmp2 and Dlx5 are regulated by Bmp4, either directly or indirectly. Mechanisms involved in the formation of sensory organs of the vertebrate inner ear are thought to be analogous to those regulating sensory bristle formation in Drosophila. Our results suggest that, in comparison to sensory bristles, crista formation within the inner ear requires an additional step of sensory and non-sensory fate specification.