Morphological Evidence for the Sensitivity of the Ear Canal of Odontocetes as shown by Immunohistochemistry and Transmission Electron Microscopy.

The function of the external ear canal in cetaceans is still under debate and its morphology is largely unknown. Immunohistochemical (IHC) analyses using antibodies specific for nervous tissue (anti-S100, anti-NSE, anti-NF, and anti-PGP 9.5), together with transmission electron microscopy (TEM) and various histological techniques, were carried out to investigate the peripheral nervous system of the ear canals of several species of toothed whales and terrestrial Cetartiodactyla. This study highlights the innervation of the ear canal with the presence of lamellar corpuscles over its entire course, and their absence in all studied terrestrial mammals. Each corpuscle consisted of a central axon, surrounded by lamellae of Schwann receptor cells, surrounded by a thin cellular layer, as shown by IHC and TEM. These findings indicate that the corpuscles are mechanoreceptors that resemble the inner core of Pacinian corpuscles without capsule or outer core, and were labelled as simple lamellar corpuscles. They form part of a sensory system that may represent a unique phylogenetic feature of cetaceans, and an evolutionary adaptation to life in the marine environment. Although the exact function of the ear canal is not fully clear, we provide essential knowledge and a preliminary hypothetical deviation on its function as a unique sensory organ.

A small lamellar corpuscle within a small nerve bundle outside the tendon of the rat soleus muscle.

A small nerve bundle outside the tendon of the adult rat soleus muscle contained a small lamellar corpuscle similar in structural organization to the ordinary paciniform corpuscle. A terminal axon composing this corpuscle was originated from a side branch of an afferent nerve fiber and surrounded by a number (approximately 15) of closely packed flattened lamellae of modified Schwann cells, while the stem nerve fiber freely terminated within the nerve bundle. These findings suggested that an afferent nerve fiber retracted after degeneration might extend a new branch within the nerve bundle and unexpectedly form a lamellar corpuscle within it.

Anatomy of the deep fascia of the upper limb. Second part: study of innervation.

Analysis of specimens taken from different areas of the deep fascia in 20 upper limbs was made in order to establish which kind of nerve fibres and endings are present in the deep muscular fascia. The flexor retinaculum and the lacertus fibrosus were also evaluated because they are anatomically hardly separable from the deep muscular fascia, although they have different functions. In particular, specimens were taken at the level of: (a) the expansion of pectoralis major onto the bicipital fascia, (b) the middle third of the brachial fascia, (c) the lacertus fibrosus, (d) the middle third of the antebrachial fascia, (e) the flexor retinaculum. This study demonstrated an abundant innervation of the fascia consisting in both free nerve endings and encapsulated receptors, in particular, Ruffini and Pacini corpuscles. However, differences in innervation were verified: the flexor retinaculum was resulted the more innervated element whilst lacertus fibrosus and the pectoralis major expansion the less innervated. These results suggest that the retinaculum has more a perceptive function whereas the tendinous expansions onto the fascia have mostly a mechanical role in the transmission of tension. The hypothesis that the fascia plays an important role in proprioception, especially dynamic proprioception, is therefore advanced. In fact, the fascia is a membrane that extends throughout the whole body and numerous muscular expansions maintain it in a basal tension. During a muscular contraction these expansions could also transmit the effect of the stretch to a specific area of the fascia, stimulating the proprioceptors in that area.

Abnormal development of pacinian corpuscles in double trkB;trkC knockout mice.

Pacinian corpuscles depend on either Aalpha or Abeta nerve fibers of the large- and intermediate-sized sensory neurons for the development and maintenance of the structural integrity. These neurons express TrkB and TrkC, two members of the family of signal transducing neurotrophin receptors, and mice lacking TrkB and TrkC lost specific neurons and the sensory corpuscles connected to them. The impact of single or double targeted mutations in trkB and trkC genes in the development of Pacinian corpuscles was investigated in 25-day-old mice using immunohistochemistry and ultrastructural techniques. Single mutations on trkB or trkC genes were without effect on the structure and S100 protein expression, and caused a slight reduction in the number of corpuscles. In mice carrying a double mutation on trkB;trkC genes most of the corpuscles were normal with a reduction of 17% in trkB-/-;trkC+/- mice, and 8% in trkB +/-;trkC -/- mice. Furthermore, a subset of the remaining Pacinian corpuscles (23% in trkB-/-;trkC+/- mice; 3% in trkB+/-;trkC-/- mice) were hypoplasic or atrophic. Present results strongly suggest that the development of a subset of murine Pacinian corpuscles is regulated by the Trk-neurotrophin system, especially TrkB, acting both at neuronal and/or peripheral level. The precise function of each member of this complex in the corpuscular morphogenesis remains to be elucidated, though.

Fine structure of myelinated mechanical nociceptor endings in cat hairy skin.

High-threshold mechanoreceptors (mechanical nociceptors) with myelinated axons were electrophysiologically identified in hairy skin of the cat as described by Burgess and Perl ('67). Such elements possess receptive fields consisting of a number of punctate areas from which maximal firing can be elicited by intense (skin-damaging) mechanical stimuli. The spots of the receptive field are separated from each other by unresponsive regions, i.e., by skin areas from which responses cannot be evoked by stimuli effective at the spots. Fine steel pins were inserted to bracket closely a number of the spotlike responsive areas for each of several units. After aldehyde perfusion of the animal, osmification of the tissue and embedding in plastic, the marked skin zones were examined in semithin and ultrathin sections at the light and electron microscopic level. Near each delineated area, a thinly myelinated axon was found that could be traced to the papillary layer where it loses its myelin sheath. Unmyelinated axons accompanied by thin Schwann cell processes were then traced and found to penetrate the epidermal basal lamina in one of the papillae. At the epidermal penetration site, the axons contained both clear round, and large, dense core vesicles; at this level, the surrounding Schwann cell cytoplasm exhibited numerous pinocytotic vesicles. The zone of penetration may constitute the receptive apparatus. Some of these axons have been traced within the basal epidermal layer where they become surrounded by keratinocytes, lose their Schwann sheath, and apparently terminate. This overall morphological pattern was consistently present in the demarked areas of focal responsiveness, and was rare in the surrounding skin; this and its difference from other cutaneous neural endings suggest that the intraepidermal axon-Schwann cel complex constitutes the receptive structure for myelinated mechanical nociceptors. It is suggested that such complexes are the sense organs responsible for initiating the sensation of pricking pain produced by localized mechanical injury of the skin.

Lectin and proteoglycan histochemistry of feline pacinian corpuscles.

We studied carbohydrate residues of glycoproteins and proteoglycans (PGs) in peritoneal Pacinian corpuscles of five adult cats. Terminal monosaccharides of glycoproteins and related polysaccharides were identified by lectin histochemistry and the PGs and glycosaminoglycans (GAGs) by specific antibodies. The most intensive lectin staining reactions indicated an abundance of glycoconjugates with terminal mannose (Man) or sialic acid residues, but no complex-type oligosaccharides were detected within the corpuscles. Terminal fucose (Fuc) and galactose (Gal) residues typical for O-linked mucin-type glycoproteins generally associated with high water binding capacity were also absent. Antibodies against unsulfated chondroitin (C-0-S), chondroitin-4-sulfate (C-4-S), and decorin showed positive reactions in the interfibrillar spaces between the lamellae, around collagen fibers, and around the lamellae of the perineural capsule, especially in the outer parts known to contain Type II collagen. Biglycan showed a preference for the innermost part of the perineural capsule (intermediate layer), known to contain Type V collagen. Collagen V and biglycan are both linked to growth processes. Hyaluronic acid (HA), chondroitin-6-sulfate (C-6-S) chains, and a chondroitin sulfate proteoglycan (CSPG) were co-localized in the terminal glia. The study of carbohydrates with high water binding capacity may contribute to our understanding of the high viscoelasticity of Pacinian corpuscles.

Ontogeny of the cutaneous sensory organs.

The ontogeny of cutaneous sensory nerve organs is described in higher vertebrates, and includes the lamellated corpuscles of Meissner, Pacini and Herbst, and the Merkel cell-neurite complex with bird Merkel and Grandry corpuscles, and mammalian Merkel cells. The main common feature is that for most corpuscles there is an inside-out order of assembly around the nerve ending which is present from the beginning of end-organ ontogeny. The exception is the mammalian Merkel cell which is present in the epidermis before the entrance of nerve fibers, and could play a promotional role in the development of skin innervation. The developmental origin of Herbst and Merkel corpuscles in birds is reported as demonstrated using embryological experiments with cell markers. Conclusions are that inner bulb cells of Herbst corpuscles and bird Merkel cells are of neural crest origin, whereas other cells (inner space and capsular cells for Herbst corpuscle and capsular cells for Merkel corpuscles) are provided by the local mesenchyme. The question of the ontogeny of mammalian Merkel cells is discussed in relation to the two debated hypothesis of epidermal and neural crest origins. Morphogenetic interactions during the development of cutaneous sensory end organs are also discussed.

Enzyme histochemistry of cutaneous sensory nerve formations.

The cutaneous sensory nerve formations belong to the structures which are studied intensely by the enzyme activity histochemistry since the early history of this technique. The histochemical localization of the activities of nonspecific cholinesterase, alkaline phosphatases, acid phosphatase, adenosine tri- and diphosphatases, adenylate cyclase, and dipeptidylpeptidase-IV in the cutaneous sensory nerve formations, mainly sensory corpuscles, is reviewed. The histochemical approach has brought new knowledge of both morphological building of these unique structures and their biochemical constituents. Taken together, the present results of enzyme histochemistry provide insight into the function of enzymes, and disclose new relationships between the sensory terminals and auxiliary structures in the cutaneous sensory nerve formations.

Role of extracellular matrix in the regeneration of a pacinian corpuscle.

The pacinian corpuscle is composed of an inner and an outer core or bulb. The former is formed by tightly packed and multi-layered thin cellular processes (lamellae) of lamellar cells which surround a centrally located axon terminal, and the latter, also called the capsule, is made up of very loosely piled layers of thin cells which encircle the inner core. Lamellar cells of the inner core are considered to be specialized Schwann cells, and the outer core cells are modified perineurial cells. In the present study, the matrix filling the extracellular spaces of the inner core consisted of basal lamina-like amorphous materials, sparce fine collagen fibrils, and the ground substance embedding these structural components. No definite basal laminae were found on the inner core lamellae except on the peripherally located ones which had distinct basal laminae. Outer-core cells were invested along the entire contour by distinct basal laminae. The interspace between the inner and outer cores was a continuation of the nerve endoneurium. The purpose of this investigation was to determine whether the extracellular matrix of the pacinian corpuscle, especially that of the inner core, has the ability to cause corpuscle regeneration, i.e. to make the regenerating axons and Schwann cells differentiate into corpuscular axon terminals and inner core cells, respectively. Pacinian corpuscles in the periosteum at the distal end of the fibula of mice were repeatedly frozen (3-5 times) in situ with forceps cooled with liquid nitrogen. Within 2-3 days, all the cellular constituents of the corpuscle had degraded, while the extracellular matrices of the inner and outer cores apparently remained undamaged. After 5-7 days, regenerating axons and accompanying immature Schwann cells entered these extracellular matrices of the inner cores. A remarkable finding was that these immature Schwann cells were detached from the axon, and sent thin cellular processes around the axon in a characteristic fashion, basically forming the same pattern as lamellae in a normal corpuscle. The regeneration of the inner core was completed by about 40 days after the freezing treatment. In the outer core, perineurial cells proliferated and extended through the basal lamina tubes of the old cells, becoming new outer core cells. These findings indicate that the extracellular matrix of the pacinian corpuscle has a specific property to cause the regeneration of the corpuscle.

Slow-adapting responses of the Pacinian corpuscles of cat planta.

Slow-adapting impulses were recorded from the single nerve fiber innervating the Pacinian corpuscle of the cat planta, which was stimulated by constant pressure. Slow-adapting Pacinian corpuscles responded to both low-frequency (0.1-0.01 Hz) and high-frequency (400-800 Hz) vibrations. Simultaneously performed electron microscopy showed that there were no definite ultrastructural differences between slow- and fast-adapting Pacinian corpuscles.

The neuroanatomical basis for the protopathic sensibility of the human glans penis.

The human glans penis is covered by stratified squamous epithelium and a dense layer of connective tissue equivalent to the dermis of typical skin. Rete ridges of the epidermis are irregular and vary in height depending on location, age, and presence or absence of a foreskin. The papillary layer of the dermis blends into and is continuous with the dense connective tissue forming the tunica albuginea of the corpus spongiosum of the glans penis. The most numerous nerve terminals are free nerve endings (FNEs) present in almost every dermal papilla, as well as scattered throughout the deeper dermis. FNEs are characterized by an incomplete Schwann cell investment, and contain irregularly scattered neurofilaments and neurotubules, clusters of mitochondria, vesicles of variable size and various inclusions. The ratio of FNEs to corpuscular receptors is approximately 10:1 and a similar ratio of small to large axons is seen in dermal nerves. Genital end bulbs are present throughout the glans, but are most numerous in the corona and near the frenulum. The unique corpuscular receptor of the glans penis consists of axon terminals that at an ultrastructural level resemble a tangled skein of FNEs. Simple, Pacinian and Ruffini corpuscles were occasionally identified predominantly in the corona glandis. Epidermal Merkel nerve endings and other types of mechanoreceptors typically found in primate glabrous skin (lip or digit) are not present. Rarely, dermal Merkel cells have been identified associated with genital end bulbs. The abundance of FNEs in isolated as well as corpuscular form can be correlated with the embryogenesis and known neurophysiologic and psychophysical parameters of sensory function of the glans penis. Finally, the divergence in reported values for the threshold of tactile and painful stimuli when applied to glabrous skin of fingertip and glans penis can be considered as an example of dissociated sensibility. The anatomical basis for this dissociation is the abundance of FNEs and absence of Merkel terminals and typical Meissner corpuscles in the covering of the glans, and the converse in glabrous skin of the digit.

Grafts of pacinian corpuscles reinnervated by dorsal root axons.

In adult rats, a piece of the crural interosseous nerve with several Pacinian corpuscles attached was removed from the crural region, autotransplanted onto the surface of the lumbar spinal cord and connected with the peripheral stump of a transected dorsal root. From 10 days up to 6 months after the operation, the grafts were investigated by light and electron microscopy. The regenerating dorsal root axons grew along the grafted nerves into the attached Pacinian corpuscles. By 1-2 months after the operation, the nerves and their branches became almost completely reinnervated by myelinated and unmyelinated dorsal root axons. In a sample of corpuscles examined 2-6 months after grafting, 75% of corpuscles were found reinnervated; each of them was supplied by 1-5 large myelinated axons that formed multiple axon terminals in the inner core. The maximal number of axonal profiles found in a transverse section through different levels of the inner core varied, in individual corpuscles, from 3 to 17 axons and terminals. The dorsal root terminals formed in the grafted corpuscles were mainly filled with mitochondria and resembled peripheral sensory endings. In some instances, the newly formed endings developed lateral processes and membrane specializations characteristic for peripheral Pacinian terminals. Thus regenerating dorsal root axons recognize a grafted peripheral mechanoreceptor as their target and reinnervate it with axon terminals, most of them structurally transformed into peripheral sensory endings.

Pacinian corpuscle in the human pancreas.

During our systematic examination of the distribution of cytochrome P450 enzymes in the normal and diseased human pancreas, we observed a Pacinian corpuscle in a serial section of a tissue from a pancreatic cancer patient. We report the histologic and immunohistochemical patterns in this corpuscle and review the literature. The Pacinian corpuscle was situated within the pancreas of a 76-year-old woman with cancer in the head of the pancreas. We could demonstrate immunoreactivity within the corpuscle for the neurofilament protein. neuron-specific enolase, S-100 Protein, and for four cytochrome P450-isozymes. The possible function of Pacinian corpuscles in the mammalian and human pancreas is discussed.

Ultrastructure of sensory nerve endings in monkey (Macaca fascicularis) knee joint capsule.

Ultrastructural studies of sensory endings in monkey posterior medial knee joint capsule were undertaken. Three distinct sensory nerve endings have been identified: free nerve endings, Ruffini corpuscles, and Pacinian corpuscles. The free nerve endings are present in all layers of the joint capsule excluding the synovium. Two types of Ruffini corpuscles have been found in the fibrous layer. The first type is characterized by a thin perineurial capsule, the second type by a thicker perineurial capsule and extensive intra-capsular space. Both types of Ruffini corpuscles are innervated by approximately one to four myelinated axons which lose their sheaths as they course through the corpuscle. They terminate on collagen fiber bundles as distinct swellings with spiny membrane projections that are covered by a thin basal lamina. These terminals contain abundant mitochondria, agranular vesicles, and irregularly arranged neurofilaments and neurotubules. Two types of Pacinian corpuscles were occasionally observed. The first was a small, typically laminated structure with an inner core at the layer between the synovium and the fibrous layer and between the fibrous layer and muscle/ligament; larger Vater-Pacinian corpuscles were noted only at the boundary between the fibrous layer and the muscle/ligament layer.

Distribution of sensory receptors in the flexor carpi radialis muscle of the cat.

The structures and distribution of encapsulated muscle receptors were examined in serial transverse sections of flexor carpi radialis in the adult cat. Four types of receptors (muscle spindles, Golgi tendon organs, paciniform, and Pacinian corpuscles) were identified. Their structures resembled those encountered in other limb muscles. Pacinian corpuscles were rare and occurred only in the external fascial coat of the muscle near its origin. The other three receptor types were distributed in an uneven but consistent pattern throughout the muscle. As noted previously (Gonyea and Ericson, '77), spindles were largely confined to a deep muscle region comprising less than 20% of the muscle volume, located directly between the long tendon of origin and the tendon of insertion. This region contains the largest proportion of type SO muscle fibers (Gonyea and Ericson, '77). Tendon organs and paciniform corpuscles were concentrated along the tendons that lined the spindle-rich muscle region. This region appeared to be composed of extrafusal fibers that were shorter and of more oblique pinnation than those in other regions. The localization of muscle receptors to the "oxidative" core of the muscle in its direct line of pull may have functional implications for afferent input to the spinal cord which are discussed. In addition, the possibility is raised that there are more paciniform corpuscles in flexor carpi radialis (and possibly other muscles) than previously thought.

Restoration of lamellar structures in adult rat Pacinian corpuscles following their simultaneous freezing injury and denervation.

The capsule and inner core are multilamellar auxiliary structures enveloping the axon terminal of the Pacinian corpuscle. The freezing injury of the rat interosseal Pacinian corpuscles induced the destruction of all cellular components while the extracellular matrix including the basal laminae survive the treatment. Simultaneous denervation and the freezing treatment of the Pacinian corpuscles discovered an ability of the basal lamina and other components of the extracellular matrix to stimulate a differentiation of migrated Schwann cells and fibroblasts into multilamellar auxiliary structures. The restoration of inner core and capsule in the Pacinian corpuscles was independent of the presence of sensory axon terminals. The restored lamellar structures of Pacinian corpuscles in long-term surviving rat (4 to 8 months) displayed atrophic changes. The results suggest that the extracellular matrix of rat Pacinian corpuscles may contain molecules that are produced by Schwann cells and fibroblasts during maturation of the multilamellar auxiliary structures. The molecules deposited into the extracellular matrix are able to influence the redifferentiation of multilamellar auxiliary structures from immature cells.

Blood-nerve barrier in the Vater-Pacini corpuscle of cat mesentery.

Correlated thin-section, freeze-fracture and tracer examinations were used to examine the blood-nerve barrier of the Vater-Pacini corpuscles in cat mesentery. A laminar inner core and a multilayered outer core enfolded the terminal nerve fiber of the corpuscle. The lamellar cells of both cores were characterized by numerous vesicular membrane invaginations. Freeze-fracture images and tracer experiments employing lanthanum nitrate proved that these invaginations are static structures mediating in neither active pinocytosis nor the transcellular transport of metabolites. In both inner and outer cores, lamellar cells were connected to one another by tight junctions of either the zonula or the fascia type, that occurred between lamellar-cell processes within the lamella and between the cells of adjacent lamellae. Intravascularly applied lanthanum lay at the outermost regions of the corpuscles without entering their internal zones, apparently because lamellar-cell tight junctions hindered further penetration. The results of our investigations suggest strongly that the Vater-Pacini corpuscle lamellae enfolding the nerve terminal form an effective diffusion barrier against the permeation of tissue fluids, thus preserving the corpuscle internal circumference.

The extracellular matrix of rat pacinian corpuscles: an analysis of its fine structure.

The Pacinian corpuscle consists of a sensory axon terminal that is enveloped by two different structures, the inner core and the capsule. Since proteoglycans are extremely water soluble and are extracted by conventional methods for electron microscopy, the current picture of the structural composition of the extracellular matrix in the inner core and the capsule of the Pacinian corpuscle is incomplete. To study the structural composition of the extracellular matrix of the Pacinian corpuscles, cationic dyes (ruthenium red, alcian blue, acridine orange) and tannic acid were applied simultaneously with the aldehyde fixation. The interosseal Pacinian corpuscles of the rat were fixed either in 2% formaldehyde and 1.5% glutaraldehyde, with the addition of one of these cationic dyes or, in Zamboni's fixative, with tannic acid added. The cationic dyes and tannic acid revealed a different structural pattern of proteoglycans in the extracellular matrix in the inner core and in the capsule of the rat Pacinian corpuscles. The inner core surrounding the sensory axon terminal is a compartment containing proteoglycans that were distributed not only in the extracellular matrix but also in the cytoplasm of the lamellae. In addition, this excitable domain was separated from the capsular fluid by a thick layer of proteoglycans on its surface. An enlarged interlamellar space of the capsule contained large amounts of proteoglycans that were removed by digestion with chondroitinase-ABC. Ruthenium red and alcian blue provided only electron dense granules, probably corresponding to collapsed monomeric proteoglycan molecules. Acridine orange and tannic acid preserved proteoglycans very well and made it possible to visualize them as "bottlebrush" structures in the electron microscope. These results show that the inner core and the capsule of rat Pacinian corpuscles have different structural patterns of proteoglycans, which are probably involved in different functions.

The electron microscopic localization of alkaline phosphatase in Pacinian corpuscles of the cat.

The ultrastructural localization of alkaline phosphatase has been studied in Pacinian corpuscles of the cat mesentery by the method of Mayahara et al. (1967) with 3 substrates. As control studies, specimens were incubated in the medium containing L-cysteine (10 mmol) or EDTA (5 mmol). The electron opaque final reaction product was observed on plasmic membranes and in cytoplasm and pinocytotic vesicles of the inner core cells. The precipitate was present also in rough endoplasmic reticulum, multivesicular bodies, and cytoplasmic vacuoles of the inner core lamellae. The axon revealed the positive enzymatic activity in the axolemma and the scattered precipitate was found in axoplasm. The pinocytotic vesicles in the capillary endothelium entering Pacinian corpuscles contained the reaction product, too. The capsule lamellae were devoid of precipitate. Localization of alkaline phosphatase in pinocytotic vesicles of the inner core lamellae and capillary wall support the opinion that this enzyme plays the significant role in the phenomenon of the transport of molecules through inner core lamellae from capillaries to the axon in Pacinian corpuscles.

Electron microscopical localization of non-specific cholinesterase activity in three principal parts of cat Pacinian corpuscles.

Non-specific cholinesterase (nCHE) activity was demonstrated histochemically on electron microscope level in 3 parts of Pacinian corpuscles from the cat mesenterium. The reaction product was abundant on the plasmic membrane of the inner core lamellae. Schwann cells of a myelinated portion of a sensory axon in Pacinian corpuscles were devoid of positive reaction to nCHE activity. These results are in agreement with those obtained in other sensory nerve endings. Therefore, the cells investing the terminal portion of an unmyelinated sensory axon are considered to be specialized Schwann cells. Furthermore, the amount of end product was lower on the specialized Schwann cells around ultraterminal than terminal portion of a sensory axon. A significantly higher nCHE activity confined to the inner core around the terminal portion of a sensory axon suggests the participation of this enzyme in the maintenance of ionic milieu in periaxonal microenvironment.