Characterization of seal whisker morphology: implications for whisker-inspired flow control applications.

Seals with beaded whiskers-the majority of true seals (Phocids)-are able to trace even minute disturbance caused by prey fish in the ambient flow using only sensory input from their whiskers. The unique three-dimensional undulating morphology of seal whiskers has been associated with their capability of suppressing vortex-induced vibration and reducing drag. The exceptional hydrodynamic traits of seal whiskers are of great interest in renovating the design of aero-propulsion flow components and high-sensitivity flow sensors. It is essential to have well-documented data of seal whisker morphology with statistically meaningful generalization, as the solid foundation for whisker-inspired flow control applications. However, the available whisker morphology data is either incomplete, with measurements of only a few key parameters, or based on a very limited sample size in case studies. This work characterizes the morphology of 27 beaded seal whiskers (harbor seal and elephant seal), using high-resolution computer-tomography scanning at NASA's Glenn Research Center in Cleveland, OH. Over two thousand cross-sectional slices for every individual whisker sample are reconstructed, to generate three-dimensional morphology. This is followed by detailed statistical analysis of a set of key parameters, under an established framework (Hanke et al 2010 J. Exp. Biol. 213 2665-72). While the length parameters are generally consistent with previous studies, we note that the angle of incidence of elliptical cross-sections varies in a wide range, with a majority falling between [Formula: see text] and [Formula: see text]. Angles of incidence at both peaks and troughs appear to roughly follow a Gaussian distribution, but no clear preference of orientation is identified. We discuss the current knowledge of whisker-inspired flow studies, focusing on choices of morphology parameters. The new understanding of whisker morphology can better inform future design of high-sensitivity flow sensors and aero-propulsion flow structures.

Ultrastructural, Sensory and Functional Anatomy of the Northern Elephant Seal (Mirounga angustirostris) Facial Vibrissae.

Vibrissae (whiskers) play a key role in underwater orientation in foraging phocids through vibrotactile sensation processing. Our aim was to evaluate the structure of northern elephant seal (NES) vibrissae by means of light (LM) and transmission electron microscopy (TEM), in order to elucidate their function. Vibrissal follicles were processed using standardized laboratory methods and LM/TEM techniques. Individual follicular axonal numbers were counted and axonal diameter measured and averaged. NES have mystacial, rhinal, supraorbital and labial vibrissae. The vibrissal follicles are histologically subdivided into a ring, upper and lower cavernous sinuses (LCS). Each vibrissa is innervated by the deep vibrissal nerve. The average number of axons per large mystacial vibrissa is 1804 (±123), rhinal 985 (±241), supraorbital 1,064 (±204) and 374 (±65) in labial vibrissa. The entire vibrissal system carries an estimated 148 573 axons, and mystacial vibrissae alone have 125 323 axons. Axonal conduction velocity for each vibrissal type is 55.26 m/s for labial, 56.58 m/s for rhinal and 35.88 m/s for mystacial vibrissae. TEM and LM revealed a plethora of mechanoreceptors within the vibrissal follicles: Merkel cell-neurite complexes, lanceolate and pilo-Ruffini end organs. A vast number of sensory axons projecting from the entire vibrissal system indicate that the vibrissal sensory area takes up a large proportion of phocids' somatosensory cortex. In conclusion, NES has highly sensitive and finely tuned vibrotactile vibrissal sense organs.

Barrelettes without barrels in the American water shrew.

Water shrews (Sorex palustris) depend heavily on their elaborate whiskers to navigate their environment and locate prey. They have small eyes and ears with correspondingly small optic and auditory nerves. Previous investigations have shown that water shrew neocortex is dominated by large representations of the whiskers in primary and secondary somatosensory cortex (S1 and S2). Flattened sections of juvenile cortex processed for cytochrome oxidase revealed clear borders of the whisker pad representation in S1, but no cortical barrels. We were therefore surprised to discover prominent barrelettes in brainstem of juvenile water shrews in the present investigation. These distinctive modules were found in the principal trigeminal nucleus (PrV), and in two of the three spinal trigeminal subnuclei (interpolaris--SpVi and caudalis--SpVc). Analysis of the shrew's whisker pad revealed the likely relationship between whiskers and barrelettes. Barrelettes persisted in adult water shrew PrV, but barrels were also absent from adult cortex. Thus in contrast to mice and rats, which have obvious barrels in primary somatosensory cortex and less clear barrelettes in the principal nucleus, water shrews have clear barrelettes in the brainstem and no barrels in the neocortex. These results highlight the diverse ways that similar mechanoreceptors can be represented in the central nervous systems of different species.

The mechanical variables underlying object localization along the axis of the whisker.

Rodents move their whiskers to locate objects in space. Here we used psychophysical methods to show that head-fixed mice can localize objects along the axis of a single whisker, the radial dimension, with one-millimeter precision. High-speed videography allowed us to estimate the forces and bending moments at the base of the whisker, which underlie radial distance measurement. Mice judged radial object location based on multiple touches. Both the number of touches (1-17) and the forces exerted by the pole on the whisker (up to 573 µN; typical peak amplitude, 100 µN) varied greatly across trials. We manipulated the bending moment and lateral force pressing the whisker against the sides of the follicle and the axial force pushing the whisker into the follicle by varying the compliance of the object during behavior. The behavioral responses suggest that mice use multiple variables (bending moment, axial force, lateral force) to extract radial object localization. Characterization of whisker mechanics revealed that whisker bending stiffness decreases gradually with distance from the face over five orders of magnitude. As a result, the relative amplitudes of different stress variables change dramatically with radial object distance. Our data suggest that mice use distance-dependent whisker mechanics to estimate radial object location using an algorithm that does not rely on precise control of whisking, is robust to variability in whisker forces, and is independent of object compliance and object movement. More generally, our data imply that mice can measure the amplitudes of forces in the sensory follicles for tactile sensation.

A mechanism of rat vibrissal movement based on actual morphology of the intrinsic muscle using three-dimensional reconstruction.

The vibrissal capsular muscle (VCM) of the rat is known to differ from the arrector pili muscle. The purpose of the present study was to characterize the rat VCM morphologically using three-dimensional reconstruction. The rat snout skin was fixed, processed with routine histological methods, sectioned serially at a thickness of 10 µm, and then stained with Masson's trichrome. The sectioned images were reconstructed three-dimensionally using 'Reconstruct' software. The findings confirmed that the VCM is a skeletal muscle attached to the vibrissal follicle such that the latter is rooted within the former. The VCM encircles the follicle almost entirely, from base to apex, and hooks around the follicle caudally. Each one of these capsular muscles is connected to two adjacent follicles in the same row. They overlap each other in the lower part, as the rostral follicular muscle that surrounds the caudal follicle. The present findings suggest that the vibrissae are able to move more freely (under voluntary control) than other general arrector pili muscles, in line with their sensory function.

Accumulated caveolae constitute subcellular compartments for glial calcium signaling in lanceolate sensory endings innervating rat vibrissae.

The terminal Schwann cells that accompany lanceolate sensory endings in the rat vibrissal follicle are known to display the small plasma membrane invaginations termed caveolae, which concentrate Ca(2+) signaling molecules. We have previously shown that these cells generate Ca(2+) signals at the lamellar processes covering the receptor axons through activation of the metabotropic purinoceptor P2Y(2). To investigate the roles of caveolae in the spatiotemporal organization of Ca(2+) signals, terminal Schwann cells were observed by immunohistochemistry for the caveola protein caveolin-1, and by transmission and scanning electron microscopy. In addition, immunohistochemical detection of P2Y(2) and its coupling partner G(q/11) along with confocal image analysis of the purinergically induced glial Ca(2+) responses was performed in isolated tissue preparations either treated or untreated with the caveolae eliminator methyl-ß-cyclodextrin. Results showed the Schwann lamellae to be characterized by the presence of dense caveolae accompanying a fine tubular network of the endoplasmic reticulum Ca(2+) store and by intense expression of the signaling molecules P2Y(2) and G(q/11). Loss of caveolae diffusely redistributed these molecules throughout the entire cell and impaired the lamellar Ca(2+) signals, both in chronological priority (preceding the global cell response) and in spatial integrity (involving the entire length of the processes). To our knowledge, this is the first report of a subcellular accumulation of caveolae underlying compartmentalized glial Ca(2+) signals that can couple with local effects on the accompanying axon terminals.

c-Maf and MafB transcription factors are differentially expressed in Huxley's and Henle's layers of the inner root sheath of the hair follicle and regulate cuticle formation.

BACKGROUND: The hair follicle of mammalian skin consists of a group of concentric epithelial cell layers. The inner root sheath (IRS), which surrounds the hardening hair shaft beneath the skin surface, is subdivided into three layers, termed the cuticle of the IRS, Huxley's layer, and Henle's layer. The IRS forms a follicular wall in the hair canal and helps guide the developing hair shaft. c-Maf and MafB, members of the Maf family of transcription factors, play important roles in the developmental processes of various tissues and in cell type-specific gene expression. OBJECTIVE: The aim of this study is to reveal the pattern of expression and functional roles of c-Maf and MafB in the hair follicle. METHODS: We determined the precise location of c-Maf and MafB expression using immunofluorescent staining of mouse skin sections with layer-specific markers. We also analyzed whiskers of c-maf- and mafB-null mice (c-maf(-/-) and mafB(-/-), respectively) using scanning electron microscopy. RESULTS: c-Maf and MafB were differentially expressed in the Huxley's and Henle's layers of the IRS. Scanning electron microscopic analysis showed irregular cuticle patterning of whiskers of c-maf(-/-) and mafB(-/-) mice. The cuticles of mafB(-/-) mice were also thinner than those of wild-type mice. CONCLUSION: c-Maf and MafB are expressed in the IRS layers in a lineage-restricted manner and are involved in hair morphogenesis.

Whisker maps in marsupials: nerve lesions and critical periods.

In the wallaby, whisker-related patterns develop over a protracted period of postnatal maturation in the pouch. Afferents arrive simultaneously in the thalamus and cortex from postnatal day (P) 15. Whisker-related patterns are first seen in the thalamus at P50 and are well formed by P73, before cortical patterns first appear (P75) or are well developed (P85). This study used the slow developmental sequence and accessibility of the pouch young to investigate the effect of nerve lesions before afferent arrival, or at times when thalamic patterns are obvious but cortical patterns not yet formed. The left infraorbital nerve supplying the whiskers was cut at P0-93 and animals were perfused at P112-123. Sections through the thalamus (horizontal plane) and cortex (tangential) were reacted for cytochrome oxidase to visualize whisker-related patterns. Lesions of the nerve at P2-5, before innervation of the thalamus or cortex, resulted in an absence of patterns at both levels. Lesions from P66-77 also disrupted thalamic and cortical patterns, despite the fact that thalamic patterns are normally well established by P73. Lesions from P82-93 resulted in normal thalamic and cortical patterns. Thus, despite the wallaby having clearly separated times for the development of patterns at different levels of the pathway, these results suggest a single critical period for the thalamus and cortex, coincident with the maturation of the cortical pattern. Possible mechanisms underpinning this critical period could include dependence of the thalamic pattern on corticothalamic activity or peripheral signals to allow consolidation of thalamic barreloids.

Microstructure and innervation of the mystacial vibrissal follicle-sinus complex in bearded seals, Erignathus barbatus (Pinnipedia: Phocidae).

Vibrissal follicle-sinus complexes (F-SCs) are sensory receptors of the mammalian integument system. They are best developed within Pinnipedia. The objective of this study was to investigate the F-SCs of bearded seals (Erignathus barbatus) for benthic foraging adaptations. Bearded seals possessed approximately 244 mystacial F-SCs. In this species, F-SCs consisted of an outer dermal capsule (DC) surrounding a blood sinus system [upper cavernous sinus (UCS), ring sinus (RS), and lower cavernous sinus (LCS)] and concentric rings of epidermal tissue. The UCS comprised up to 62% of the F-SC length and may function as thermal protection for mechanoreceptors. A large asymmetrical ringwulst was located in the RS. A deep vibrissal nerve penetrated the DC at its base and terminated on mechanoreceptors in the epidermal tissues of the LCS and RS. The mean number of myelinated axons per F-SC was 1,314 (range, 811-1,650) and was among the highest number of axons per F-SC reported to date. An estimated mean number of 320,616 myelinated axons innervate the entire mystacial vibrissal array. Merkel-Neurite complexes (MNCs) and small simple laminated corpuscles were found in the region of the LCS. Myelinated axons also terminated on MNCs and lanceolate endings apical to the ringwulst. The number of F-SCs, their geometry in the mystacial region, the number of myelinated axons per F-SC, and the distribution of mechanoreceptors support the premise that pinniped vibrissae are sensitive active-touch receptor systems, and that structural differences in bearded seals, relative to other phocids, may be adaptations for benthic foraging.

Targeted ablation of the abcc6 gene results in ectopic mineralization of connective tissues.

Pseudoxanthoma elasticum (PXE), characterized by connective tissue mineralization of the skin, eyes, and cardiovascular system, is caused by mutations in the ABCC6 gene. ABCC6 encodes multidrug resistance-associated protein 6 (MRP6), which is expressed primarily in the liver and kidneys. Mechanisms producing ectopic mineralization as a result of these mutations remain unclear. To elucidate this complex disease, a transgenic mouse was generated by targeted ablation of the mouse Abcc6 gene. Abcc6 null mice were negative for Mrp6 expression in the liver, and complete necropsies revealed profound mineralization of several tissues, including skin, arterial blood vessels, and retina, while heterozygous animals were indistinguishable from the wild-type mice. Particularly striking was the mineralization of vibrissae, as confirmed by von Kossa and alizarin red stains. Electron microscopy revealed mineralization affecting both elastic structures and collagen fibers. Mineralization of vibrissae was noted as early as 5 weeks of age and was progressive with age in Abcc6(-/-) mice but was not observed in Abcc6(+/-) or Abcc6(+/+) mice up to 2 years of age. A total body computerized tomography scan of Abcc6(-/-) mice revealed mineralization in skin and subcutaneous tissue as well as in the kidneys. These data demonstrate aberrant mineralization of soft tissues in PXE-affected organs, and, consequently, these mice recapitulate features of this complex disease.

Reduced plasticity of cortical whisker representation in adult tenascin-C-deficient mice after vibrissectomy.

The effect of the extracellular matrix recognition molecule tenascin-C on cerebral plasticity induced by vibrissectomy was investigated with 2-deoxyglucose (2DG) brain mapping in tenascin-C-deficient mice. Unilateral vibrissectomy sparing row C of vibrissae was performed in young adult mice. Two months later, cortical representations of spared row C vibrissae and control row C on the other side of the snout were visualized by [(14)C]2DG autoradiography. In both wild-type and tenascin-C-deficient mice, cortical representation of the spared row was expanded in all layers of the barrel cortex. However, the effect was significantly more extensive in wild-type animals than in the mutant. Elimination of tenascin-C by genetic manipulation thus reduces the effect of vibrissectomy observed in the somatosensory cortex. No increase in number of fibres in the vibrissal nerve of spared vibrissae was seen, and occurrence of additional nerve to the spared follicle was very rare. Thus, in tenascin-C-deficient mice functional plasticity seems to be impaired within the CNS.

The scanning electron and light microscopic structure of bovine tactile hair.

Bovine tactile hairs in skin samples from the lateral side of the upper lip were examined using scanning electron and light microscopy. The root of these hairs has a variable length and is surrounded by a large sinus located between the internal and the external dermal sheath. With a prominent thickness, the external dermal sheath forms the external wall of the tactile hair and contains many nerves some of which extend into trabeculae. Trabeculae projecting from the internal dermal sheath and attaching to the external dermal sheath with two or more branches are present in the entire sinus. The trabeculae are interconnected by connective tissue sheets that support the integrity of the trabecular organization. The sinus surfaces as well as trabeculae are lined by endothelia. As a result, the bovine tactile hair is truly a cavernous type of tactile hair with a well organized microscopic anatomy. Thus, the bovine tactile hair most likely plays an important role in relatively immobile and insensitive bovine lips.

Scanning electron microscopic study of different hair types in various breeds of rabbits.

The microarchitecture of the cover hairs, wool hairs and tactile (sinus) hairs of feral, New Zealand White and Angora rabbits was studied by means of scanning electron microscopy. The morphology and variability of the cuticular scale patterns, hair cortex, medullary arrangement and profile of the hairs are described, illustrated and compared with findings resulting from conventional light microscopy, cuticular casting and medullary impregnation. All parameters examined in cover hairs presented a considerable variation along the length of the hair shaft. In wool hairs, in contrast, only the cuticular scale pattern was subject to manifest segmental variation, whereas the shaft diameter, cortical profile and medullar composition changed little over the entire length of the hair. The tactile hairs of the head were characterised by a round profile of the hair shaft, a cylindrical central medullar canal, and a thick cortex covered by cuticular scales that were arranged in a waved pattern and oriented transversally in relation to the longitudinal axis of the hair. It was concluded that the scanning electron microscopic observation of hair samples is a fast and valuable method for identifying hair types with useful applications in different disciplines such as mammalian biology, the textile industry and forensic medicine.

Microanatomy of facial vibrissae in the Florida manatee: the basis for specialized sensory function and oripulation.

Sirenians, including Florida manatees, possess an array of hairs and bristles on the face. These are distributed in a pattern involving nine distinct regions of the face, unlike that of any other mammalian order. Some of these bristles and hairs are known to be used in tactile exploration and in grasping behaviors. In the present study we characterized the microanatomical structure of the hair and bristle follicles from the nine regions of the face. All follicles had the attributes of vibrissae, including a dense connective tissue capsule, prominent blood sinus complex, and substantial innervation. Each of the nine regions of the face exhibited a distinct combination of these morphological attributes, congruent with the previous designation of these regions based on location and external morphological criteria. The present data suggest that perioral bristles in manatees might have a tactile sensory role much like that of vibrissae in other mammals, in addition to their documented role in grasping of plants during feeding. Such a combination of motor and sensory usages would be unique to sirenians. Finally, we speculate that the facial hairs and bristles may play a role in hydrodynamic reception.

Three-dimensional microanatomy of longitudinal lanceolate endings in rat vibrissae.

The longitudinal lanceolate endings are ubiquitous sensory terminals in the sinus and nonsinus hairs of mammals that form a palisade around the hair follicle. To analyze how the nerve endings detect hair movements, the present study re-examined their fine structure and relationships with surrounding connective tissue in rat vibrissae by using a combination of three methods: immunohistochemistry for S-100 protein, scanning electron microscopy of NaOH-macerated specimens, and transmission electron microscopy of serial sections. Observations showed the lanceolate endings to be represented by triplet units with a flattened axon terminal flanked on each side by a Schwann cell lamella, as reported previously. Two distinct parts were discriminated in the lanceolate ending: a principal portion in which the axon terminal protruded numerous fine fingers from between the Schwann cell coverings, and an apical cone that enclosed a large axon finger in an attenuated Schwann sheath. Long foot processes of Schwann cells fanned out distally from each apical cone. The principal portions of the lanceolate endings were firmly linked to the surrounding connective tissue by the narrow edges equipped with axon fingers, suggesting their continuous deformation by sustained hair deflections. In contrast, the apical cones were freely suspended in an amorphous matrix with only the end feet of the Schwann cell projections attached to rigid tissue elements. This part of the ending was proposed as a possible transducer site to generate rapidly adapting receptor potentials, both retreating and overshooting during the acceleration and deceleration phases of a given vibrissal movement.

Partial denervation of the whiskerpad in adult mice: pattern and origin of reinnervation.

We studied sensory organ reinnervation after nerve transection in the mouse whisker-to-barrel pathway. In one set of adult mice, we determined at light microscopy level the number of fibres reaching the caudal whisker follicles 5, 15, 20, 60, 100 days and 1 year after transection of the sensory nerve of row C. Regenerated fibres were first detected 15 days post lesionem (p.l.) and myelin first observed at 20 days. Between 60 and 100 days, the number of fibres stayed at approximately 80% of the values obtained in control animals. At that time, myelinated fibres reached only 58% of their number in controls. At the electron microscopy level, these fibres differ from control ones by a smaller fibre diameter. The innervation of follicles of adjacent rows was not modified, indicating that follicular reinnervation is row specific. We checked this feature by injecting in another set of mice the denervated follicles and the adjacent ones with distinct retrograde tracers 45 days and 1 year after nerve transection. The percentage of double-labelled neurons in the Gasserian ganglion did not increase in experimental animals. This confirms the absence of colonization of intact follicles by regenerating fibres and indicates that reinnervation of the whisker follicles takes place by regeneration of the degenerated axons without collateral reinnervation. The companion paper describes the pattern of activation of the barrel cortex relative to the present findings.

Ectopic expression of the nude gene induces hyperproliferation and defects in differentiation: implications for the self-renewal of cutaneous epithelia.

Nude mice are characterized by the absence of visible hair, epidermal defects, and the failure to develop a thymus. This phenotype results from loss-of-function mutations in Whn (Hfh11), a winged-helix transcription factor. In murine epidermis and hair follicles, endogenous whn expression is induced as epithelial cells initiate terminal differentiation. Using the promoter for the differentiation marker involucrin, transgenic mice that ectopically express whn in stratified squamous epithelia, hair follicles, and the transitional epithelium of the urinary tract were generated. Transgenic epidermis and hair follicles displayed impaired terminal differentiation and a subset of hair defects, such as delayed growth, a waved coat, and curly whiskers, correlated with decreased transforming growth factor (TGF)-alpha expression. The exogenous Whn protein also stimulated epithelial cell multiplication. In the epidermis, basal keratinocytes exhibited hyperproliferation, though transgene expression was restricted to suprabasal, postmitotic cells. Hair follicles failed to enter telogen (a resting period) and remained continuously in an abnormal anagen (the growth phase of the hair cycle). Ureter epithelium developed severe hyperplasia, leading to the obstruction of urine outflow and death from hydronephrosis. Though an immune infiltrate was present occasionally in transgenic skin, the infiltrate was not the primary cause of the epithelial hyperproliferation, as the immune reaction was not observed in all affected transgenics, and the transgene induced identical skin and urinary tract abnormalities in immunodeficient Rag1-null mice. Given the effects of the transgene on cell proliferation and TGFalpha expression, the results suggest that Whn modulates growth factor production by differentiating epithelial cells, thereby regulating the balance between proliferative and postmitotic populations in self-renewing epithelia.

Ultrastructural observations of synaptic connections of vibrissa afferent terminals in cat principal sensory nucleus and morphometry of related synaptic elements.

Previous work suggests that slowly adapting (SA) periodontal afferents have different synaptic arrangements in the principal (Vp) and oral trigeminal nuclei and that the synaptic structure associated with transmitter release may be related directly to bouton size. The present study examined the ultrastructures of SA and fast adapting (FA) vibrissa afferents and their associated unlabeled axonal endings in the cat Vp by using intra-axonal labeling with horseradish peroxidase and a morphometric analysis. All SA and FA afferent boutons contained clear, round, synaptic vesicles. All the FA and most SA boutons were presynaptic to dendrites, but a few SA boutons were axosomatic. Both types of bouton were frequently postsynaptic to unlabeled axonal ending(s) containing pleomorphic, synaptic vesicles (P-ending). The size of labeled boutons was larger in FA than SA afferents, but the size of dendrites postsynaptic to labeled boutons was larger for SA than FA afferents. Large-sized FA and SA boutons made synaptic contacts with small-diameter dendrites. The size of FA and SA boutons was larger than that of their associated P-endings. A morphometric analysis made on the pooled data of SA and FA boutons indicated that apposed surface area, active zone number, total active zone area, vesicle number, and mitochondrial volume were highly correlated in a positive linear manner with labeled bouton volume. These relationships were also applicable to unlabeled P-endings, but the range of each parameter was smaller than that of the labeled boutons. These observations provide evidence that the two functionally distinct types of vibrissa afferent manifest unique differences but share certain structural features in the synaptic organization and that the ultrastructural "size principle" proposed by Pierce and Mendell ([1993] J. Neurosci. 13:4748-4763) for Ia-motoneuron synapses is applicable to the somatosensory system.