E. H. Peterson

Computational models of hair cell bundle mechanics: III. 3-D utricular bundles

Six utricular hair bundles from a red-eared turtle are modeled using 3-D finite element analysis. The mechanical model includes shear deformable stereocilia, realignment of all forces during force load increments, and tip and lateral link inter-stereocilia connections. Results show that there are two distinct bundle types that can be separated by mechanical bundle stiffness. The more compliant group has fewer total stereocilia and short stereocilia relative to kinocilium height; these cells are located in the medial and lateral extrastriola. The stiff group are located in the striola. They have more stereocilia and long stereocilia relative to kinocilia heights. Tip link tensions show parallel behavior in peripheral columns of the bundle and serial behavior in central columns when the tip link modulus is near or above that of collagen (1x10(9) N/m(2)). This analysis shows that lumped parameter models of single stereocilia columns can show some aspects of bundle mechanics; however, a distributed, 3-D model is needed to explore overall bundle behavior.

Kinocilia heights on utricular hair cells.

Kinocilium height is a critical determinant of any hair cells response to head movement, but accurate measurements of kinocilia heights have been difficult to achieve. We have developed a method for measuring kinocilia heights that combines immunochemical staining with three-dimensional morphometry, and we have used this method to measure kinocilia in the utricle of a turtle, Pseudemys scripta. Our results suggest that kinocilium height varies with position on the utricular epithelium and that kinocilia in the striola are significantly shorter than kinocilia in other regions of the utricle.

Steady-state stiffness of utricular hair cells depends on macular location and hair bundle structure

Spatial and temporal properties of head movement are encoded by vestibular hair cells in the inner ear. One of the most striking features of these receptors is the orderly structural variation in their mechanoreceptive hair bundles, but the functional significance of this diversity is poorly understood. We tested the hypothesis that hair bundle structure is a significant contributor to hair bundle mechanics by comparing structure and steady-state stiffness of 73 hair bundles at varying locations on the utricular macula. Our first major finding is that stiffness of utricular hair bundles varies systematically with macular locus. Stiffness values are highest in the striola, near the line of hair bundle polarity reversal, and decline exponentially toward the medial extrastriola. Striolar bundles are significantly more stiff than those in medial (median: 8.9 μN/m) and lateral (2.0 μN/m) extrastriolae. Within the striola, bundle stiffness is greatest in zone 2 (106.4 μN/m), a band of type II hair cells, and significantly less in zone 3 (30.6 μN/m), which contains the only type I hair cells in the macula. Bathing bundles in media that break interciliary links produced changes in bundle stiffness with predictable time course and magnitude, suggesting that links were intact in our standard media and contributed normally to bundle stiffness during measurements. Our second major finding is that bundle structure is a significant predictor of steady-state stiffness: the heights of kinocilia and the tallest stereocilia are the most important determinants of bundle stiffness. Our results suggest 1) a functional interpretation of bundle height variability in vertebrate vestibular organs, 2) a role for the striola in detecting onset of head movement, and 3) the hypothesis that differences in bundle stiffness contribute to diversity in afferent response dynamics.

Utricular afferents: morphology of peripheral terminals

The utricle provides critical information about spatiotemporal properties of head movement. It comprises multiple subdivisions whose functional roles are poorly understood. We previously identified four subdivisions in turtle utricle, based on hair bundle structure and mechanics, otoconial membrane structure and hair bundle coupling, and immunoreactivity to calcium-binding proteins. Here we ask whether these macular subdivisions are innervated by distinctive populations of afferents to help us understand the role each subdivision plays in signaling head movements. We quantified the morphology of 173 afferents and identified six afferent classes, which differ in structure and macular locus. Calyceal and dimorphic afferents innervate one striolar band. Bouton afferents innervate a second striolar band; they have elongated terminals and the thickest processes and axons of all bouton units. Bouton afferents in lateral (LES) and medial (MES) extrastriolae have small-diameter axons but differ in collecting area, bouton number, and hair cell contacts (LES >> MES). A fourth, distinctive population of bouton afferents supplies the juxtastriola. These results, combined with our earlier findings on utricular hair cells and the otoconial membrane, suggest the hypotheses that MES and calyceal afferents encode head movement direction with high spatial resolution and that MES afferents are well suited to signal three-dimensional head orientation and striolar afferents to signal head movement onset.

Quantifying utricular stimulation during natural behavior.

The use of natural stimuli in neurophysiological studies has led to significant insights into the encoding strategies used by sensory neurons. To investigate these encoding strategies in vestibular receptors and neurons, we have developed a method for calculating the stimuli delivered to a vestibular organ, the utricle, during natural (unrestrained) behaviors, using the turtle as our experimental preparation. High-speed digital video sequences are used to calculate the dynamic gravito-inertial (GI) vector acting on the head during behavior. X-ray computed tomography (CT) scans are used to determine the orientation of the otoconial layer (OL) of the utricle within the head, and the calculated GI vectors are then rotated into the plane of the OL. Thus, the method allows us to quantify the spatio-temporal structure of stimuli to the OL during natural behaviors. In the future, these waveforms can be used as stimuli in neurophysiological experiments to understand how natural signals are encoded by vestibular receptors and neurons. We provide one example of the method, which shows that turtle feeding behaviors can stimulate the utricle at frequencies higher than those typically used in vestibular studies. This method can be adapted to other species, to other vestibular end organs, and to other methods of quantifying head movements.

A novel machine vision application for analysis and visualization of confocal microscopic images

Abstract.In this paper, a novel machine vision application is presented for analyzing and visualizing confocal microscopy images of biological preparations. The proposed system is divided into three subsystems: a 3D curved surface extraction subsystem that generates 3D surfaces passing through selected key points in confocal image stacks; a 2D image projection subsystem that produces a flattened projection of the extracted curved surface; and an image mosaic subsystem that concatenates a series of image projections to form a view of an entire biological preparation. A combination of cubic interpolation and boundary matching is employed to reconstruct the 3D curved surface that passes through selected key points. The projection process integrates data fidelity and local smoothness constraints, producing a color or intensity projection along the desired 3D surface. Registration is achieved by aligning and minimizing the sum of the squared distances (SSD) between the intensities of the corresponding pixels. Two biological applications of the proposed system are reported to illustrate how the vision system could aid in biological research.

Chapter 21 Neuromuscular Strategies Underlying Ballistic Movements

Publisher Summary The major goal of motor control research is to advance the understanding of the central nervous system (CNS) mechanisms that bring about movement diversity. Such diversity results from two sources of variation: (1) the design of musculoskeletal elements that participate in movement and (2) the way these elements are activated by the CNS. Conceptually, the simplest approach to understanding how a neuromuscular system might generate a specific type of movement is to begin by studying the individual properties of its various components—for example, the muscle fiber (MF), the motor unit (MU), and the motoneurons (MNs). Eventually, a picture begins to emerge showing how the distinctive components might function during movement. For example, skeletal muscles are composed of MFs that have different morphological (e.g., lengths, diameters, and tapering profiles), physiological, and biochemical (e.g., contraction speed and fatigability) properties. Each of these properties and their various combinations confer certain capabilities on the muscle. The assembly of individual MFs with different properties to form whole MUs and whole muscles and the activation of this assembly of usually heterogeneous MFs and MUs by the CNS, at both segmental and supra-segmental levels, are some of the important questions to be addressed. This approach has been applied with great success to the groups of muscles that are involved in the cyclical patterns of muscle activation that are driven by central pattern-generating circuits (e.g., chewing, scratching, and locomotion). In contrast, much less is known about muscle systems that are involved in ballistic movements. This chapter discusses the design of the various musculoskeletal and neural elements that participate in ballistic head retraction—the chief escape response in turtles.

Models of utricular bouton afferents: role of afferent-hair cell connectivity in determining spike train regularity

Vestibular bouton afferent terminals in turtle utricle can be categorized into four types depending on their location and terminal arbor structure: lateral extrastriolar (LES), striolar, juxtastriolar, and medial extrastriolar (MES). The terminal arbors of these afferents differ in surface area, total length, collecting area, number of boutons, number of bouton contacts per hair cell, and axon diameter (Huwe JA, Logan CJ, Williams B, Rowe MH, Peterson EH. J Neurophysiol 113: 2420-2433, 2015). To understand how differences in terminal morphology and the resulting hair cell inputs might affect afferent response properties, we modeled representative afferents from each region, using reconstructed bouton afferents. Collecting area and hair cell density were used to estimate hair cell-to-afferent convergence. Nonmorphological features were held constant to isolate effects of afferent structure and connectivity. The models suggest that all four bouton afferent types are electrotonically compact and that excitatory postsynaptic potentials are two to four times larger in MES afferents than in other afferents, making MES afferents more responsive to low input levels. The models also predict that MES and LES terminal structures permit higher spontaneous firing rates than those in striola and juxtastriola. We found that differences in spike train regularity are not a consequence of differences in peripheral terminal structure, per se, but that a higher proportion of multiple contacts between afferents and individual hair cells increases afferent firing irregularity. The prediction that afferents having primarily one bouton contact per hair cell will fire more regularly than afferents making multiple bouton contacts per hair cell has implications for spike train regularity in dimorphic and calyx afferents.NEW & NOTEWORTHY Bouton afferents in different regions of turtle utricle have very different morphologies and afferent-hair cell connectivities. Highly detailed computational modeling provides insights into how morphology impacts excitability and also reveals a new explanation for spike train irregularity based on relative numbers of multiple bouton contacts per hair cell. This mechanism is independent of other proposed mechanisms for spike train irregularity based on ionic conductances and can explain irregularity in dimorphic units and calyx endings.

Afferent-hair cell connectivity as a possible source of spike train irregularity in turtle vestibular bouton afferents

Vestibular afferents have been categorized as “regular” or “irregular” depending on the variability of their spike trains [1,2]. It is thought that the difference in regularity among afferents results from differences in the types of voltage-dependent conductances present in the cell. For example afferents may differ in their complements of potassium A, D, and M currents or in currents that produce a small vs. prominent afterhyperpolarization (AHP) [3-5]. Because bouton afferents from turtle utricle vary widely in morphology depending on their location in the utricle, we sought to determine if afferent morphology might also play a role in spike train irregularity. Although we found that morphology itself is not a factor in determining spike train variability, we did find that spike train variability can be affected by afferent/hair cell connectivity. Morphological reconstructions of bouton afferents from the lateral extrastriolar (LES), striola, juxtastriola and medial extrastriolar (MES) regions of turtle utricle were done with Neurolucida. Models based on these reconstructions were constructed for 16 afferents. Voltage-dependent conductance distributions in different regions (axon, soma, terminal arbor, varicosities, nodes, myelin) were identical in each cell model. Given the locations of synapses in the terminal arbor and the density of hair cells in each region, we were able to estimate the number of synapses formed between individual hair cells and an afferent. In the LES, striola and juxtastriola most synapses on an afferent were formed with unique hair cells, whereas in the MES single hair cells more often made multiple synapses with an afferent. To determine the effect of this difference in connectivity we modeled Poisson release from hair cells to synapses in the terminal arbor to generate spike trains. The coefficient of variation (cv) as a function of interspike interval (ISI) was calculated to estimate spike train variability with different average release rates. We found that cv as a function of ISI was nearly identical for LES, striola and juxtastriola afferents, but was the same for MES afferents only if connectivity in the MES was assumed to be one afferent synapse per hair cell. However, cv was much larger when the MES afferents were modeled with multiple synapses between an afferent and a hair cell, as calculated from synaptic location and hair cell density. To see if we could make an LES afferent more irregular, we modified connectivity to have more multiple synapses between individual hair cells and the afferent. As predicted, cv increased. These results suggest that afferent-hair cell connectivity may be a source of spike train irregularity. Given the large number of multiple contacts between a calyx and its presynaptic hair cell, we expect that connectivity contributes to spike train variability in calyx-bearing (calyx and dimorph) afferents.

Signal processing in posterior-canal bouton vestibular primary afferents

Empirical studies show that the discharge statistics and response dynamics of vestibular afferents differ depending on the type and location of the hair cells they innervate. In the turtle, the type of hair cell and structure of innervating afferents vary depending on sensory surface location; type I and II hair cells are found in the central zone (CZ) and are innervated by calyx (C), dimorphic (D) and bouton (B) afferents, while only type II hair cells innervated by bouton afferents are present in the peripheral zone (PZ). Bouton afferents located near the canal wall (BP) have regular discharge statistics, and have small gains and phase leads with respect to angular head velocity, whereas B units close to the canal center (BT) have irregular discharges and have large gain and phase leads. Calyx and dimorphic units both have irregular discharge patterns and intermediate gain and phase leads. Perhaps because of their unusual morphology, considerable attention has been paid to signal processing by calyces. However, few studies have examined hair cell-to-afferent signaling in bouton afferents, even though they are the only terminal type found in fish and amphibians and they are a significant population in the semicircular canals of mammals, reptiles and birds. We are studying signal processing in bouton afferents from the peripheral zone of the posterior canal of the turtle, in particular the contrast between BP and BT cells. We have to date obtained morphological reconstructions of two intra-axonally filled electrophysiologically characterized cells, one BT and one BP unit, that differed substantially in their morphology; the BT unit had 60% more boutons than the BP unit (40 vs 25), it had a larger collection area (5000 vs 3000 μm2) and a larger projection along the wall to center axis (125 vs 60 mm). These results are consistent with previous reports. We used the above morphologies to construct multi-compartmental models in NEURON. At present, we assume one synapse per bouton. We adjusted independent Poisson vesicle release rates and synaptic conductances to match experimental reports, and incorporated fNa and KDR type voltage-gated channels in axonal compartments. Preliminary results under resting conditions indicate firing patterns consistent with experimental reports in terms of firing rates and the CV of interspike intervals. Preliminary results also indicate that terminal morphology has a measurable influence on discharge regularity as measured by the CV of interspike interval distributions, but the differences are smaller than the measured differences for these units, suggesting that other variables (e.g., number of synapses/bouton; channel kinetics) interact with morphology to shape discharge statistics. Simulations of dynamic behavior are in progress. from Seventeenth Annual Computational Neuroscience Meeting: CNS*2008 Portland, OR, USA. 19–24 July 2008