In vitro formation of the Merkel cell‐neurite complex in embryonic mouse whiskers using organotypic co‐cultures

A Merkel cell‐neurite complex is a touch receptor composed of specialized epithelial cells named Merkel cells and peripheral sensory nerves in the skin. Merkel cells are found in touch‐sensitive skin components including whisker follicles. The nerve fibers that innervate Merkel cells of a whisker follicle extend from the maxillary branch of the trigeminal ganglion. Whiskers as a sensory organ attribute to the complicated architecture of the Merkel cell‐neurite complex, and therefore it is intriguing how the structure is formed. However, observing the dynamic process of the formation of a Merkel cell‐neurite complex in whiskers during embryonic development is still difficult. In this study, we tried to develop an organotypic co‐culture method of a whisker pad and a trigeminal ganglion explant to form the Merkel cell‐neurite complex in vitro. We initially developed two distinct culture methods of a single whisker row and a trigeminal ganglion explant, and then combined them. By dissecting and cultivating a single row from a whisker pad, the morphogenesis of whisker follicles could be observed under a microscope. After the co‐cultivation of the whisker row with a trigeminal ganglion explant, a Merkel cell‐neurite complex composed of Merkel cells, which were positive for both cytokeratin 8 and SOX2, Neurofilament‐H‐positive trigeminal nerve fibers and Schwann cells expressing Nestin, SOX2 and SOX10 was observed via immunohistochemical analyses. These results suggest that the process for the formation of a Merkel cell‐neurite complex can be observed under a microscope using our organotypic co‐culture method.     

THE INTRAEPIDERMAL INNERVATION OF THE SNOUT SKIN OF THE OPOSSUM : A Light and Electron Microscope Study, with Observations on the Nature of Merkel's Tastzellen

The intraepidermal innervation of the snout skin of the opossum has been studied with the light and electron microscope. Numerous large nerve fibers loose their myelin sheath in the superficial dermis and pass into the epidermis. The basement membranes of the epidermis and Schwann cell become continuous at the point of entry of the neurite into the epidermis. Within the epidermis, the neurite is associated with a specialized secretory epidermal cell, termed a Merkel cell. This cell has many secretory granules apposed to the neurite. The Merkel cells are epidermal cells since they have desmosomes between them and adjacent epidermal cells. The neurite in the stratum spinosum is enveloped by Schwann cells in a manner analogous to the Schwann cell investment of unmyelinated neurites. In the upper stratum spinosum the nerve fiber evidences changes which can be interpreted as degenerative. The Merkel cell-neurite complex is interpreted as representing a sensory receptor unit.     

Evidence of fast serotonin transmission in frog slowly adapting type 1 responses

The Merkel cell–neurite (MCN) complex generates slowly adapting type 1 (SA1) response when mechanically stimulated. Both serotonin (5-HT) and glutamate have been implicated in the generation of normal SA1 responses, but previous studies have been inconclusive as to what their roles are or how synaptic transmission occurs. In this study, excised dorsal skin patches from common water frogs (Rana ridibunda) were stimulated by von Frey hairs during perfusion in a tissue bath, and single-unit spike activity was recorded from SA1 fibres. Serotonin had no significant effect on the SA1 response at low (10?µM) concentration, significantly increased activity in a force-independent manner at 100?µM, but decreased activity with reduced responsiveness to force at 1?mM. Glutamate showed no effect on the responsiveness to force at 100?µM. MDL 72222 (100?µM), an ionotropic 5-HT₃ receptor antagonist, completely abolished the responsiveness to force, suggesting that serotonin is released from Merkel cells as a result of mechanical stimulation, and activated 5-HT₃ receptors on the neurite. The metabotropic 5-HT₂ receptor antagonist, ketanserin, greatly reduced the SA1 fibre's responsiveness to force, as did the non-specific glutamate receptor antagonist, kynurenic acid. This supports a role for serotonin and glutamate as neuromodulators in the MCN complex, possibly by activation and/or inhibition of signalling cascades in the Merkel cell associated with vesicle release. Additionally, it was observed that SA1 responses contained a force-independent component, similar to a dynamic response observed during mechanical vibrations.     

The fine structure of the lateral eyes ofNeocarus texanus Chamberlin and Mulaik, 1942 (Opilioacarida,Acari, Arachnida,Chelicerata)

Summary Neocarus texanus, a primitive mite, bears two pairs of eyes, which are principally similar in ultrastructure. Each eye is covered externally by a cuticular cornea. It is underlain by flat sheath cells which send extensive processes into the retina. The retina is composed of distal and proximal cells. The 20 distal cells of the anterior eye are inversely orientated and form 10 disc-like rhabdoms. They represent typical retinula cells. Each rhabdom encloses the dendritic process of a neuron, the perikaryon of which is located outside the retina (proximal cells). The significance of this cell is not known. The retina is underlain by a crystalline tapetum. In the posterior eye 14 retinula cells form 7 rhabdoms in an arrangement similar to the anterior eye. The eyes of one side of the body are located within a capsule of pigment cells. Together the axons of the distal and proximal cells form the two optic nerves, one on each side of the body. The optic nerves leave the eyes anteriorly and terminate in two optic neuropils located in the brain.From structural evidence it is concluded, that the resolution of the eyes must be rather low.The peculiar proximal cells have not been observed previously in Acari. They probably resemble at best the eccentric cells and arhabdomeric cells of xiphosurans, scorpions, whip-scorpions and opilionids. Also, inverse retinae and tapeta of the present type have not been found in Acari until now, but are present in other Arachnida. Thus the eyes ofNeocarus texanus evidently represent a unique type within the Acari.     

Ultrastructure of the sensory palps ofTetranchyroderma papii (Gastrotricha,Macrodasyida)

Summary The sensory palps of the macrodasyoid gastrotrichTetranchyroderma papii contain processes from two types of cell: 22–23 bipolar primary sensory cells and two to three support cells. In the proximal region of the palp each sensory cell contains a short ciliary segment with a basal body and from this ciliary segment a longer distal segment lacking axonemal microtubules extends through the major part of the length of the palp. Each support cell process bears microvilli and contains a conspicuous bundle of microtubules running the entire length of the process. The cell bodies of both cell types are situated in the epidermis of the head region. The palps are interpreted as having a chemosensory function. They are considered to be homologous to the posterior cephalic sensory organ ofTurbanella cornuta, but not the head tentacles ofChordodasys antennatus or nematode amphids.     

Structure and development of the nephridia of Tomopteris helgolandica (Annelida)

 Nephridial diversity is high in Phyllodocida (Annelida) and ranges from protonephridia to metanephridia. The nephridia of Tomopteris helgolandica (Tomopteridae) can be characterized as metanephridia which bear a multiciliated solenocyte. This cell is medially apposed to the proximal part of the nephridial duct and bears several cilia, each of which is surrounded by a ring of 13 microvilli. An extracellular matrix connects the microvilli and thus leads to the impression of a tube surrounding the central cilium. Each tube separately enters a subjacent duct cell and the cilia extend into a cup-shaped compartment within the duct cell. This compartment is not connected to the duct. The funnel consists of eight multiciliated cells and is connected to the nephridial duct, which initially runs intercellularly and later percellularly. The last duct cell bears a neck-like process which pierces the subepidermal basal membrane and is connected to epidermal cells forming a small invagination, the nephropore. The nephridia of T. helgolandica develop from a band of cells and all structural components are differentiated at an early developmental stage. Further development is characterized by enlargment of the funnel, ciliogenesis in the solenocyte, merging of different sections of the duct and, finally, the formation of the nephropore. An evaluation of the nephridia of T. helgolandica leads to the hypothesis that the nephridial diversity in Phyllodocida can be explained by the retainment of different stages in the transition of protonephridia into metanephridia; this is caused by the formation of a ciliated funnel at different ontogenetic stages. Although the protonephridia in Phyllodocida are regarded as primary nephridial organs, protonephridia are also presumed to have evolved secondarily in progenetic interstitial species of the Annelida by an incomplete differentiation of the nephridial anlage. Accepted: 18 December 1996     

On the occurrence of merkel cells in the epidermis of teleost fishes

Summary The ultrastructure of a differentiated cell type in the epidermis of two species of teleost fish, Ictalurus melas and Phoxinus phoxinus, is described. This cell type has a synaptic association with nerve fibres, microvillus-like peripheral processes, and membrane-bounded inclusions, which together are the diagnostic features of the Merkel cells of tetrapod vertebrates. Other cytoplasmic features are shared with the epithelial cells. The appearance of the membrane-bounded granules depends on the fixative used; after fixation with glutaraldehyde the granules are of a size and electron-density comparable to that found in tetrapod Merkel cells, but after fixing in osmium tetroxide the granules are inconspicuous.Our thanks are due to Mr. A.C. Wheeler of the British Museum (Natural History) for help with the identification of the species of Ictalurus, and to Mr. E. Perry for technical assistance. One author (EBL) was supported by a SRC research studentship     

Ultrastructure of Merkel corpuscles in the tongue of the finch,Lonchura striata

Summary Merkel corpuscles in the lingual mucosa of the finch, Lonchura striata, were examined by means of the argyrophilic reaction and electron microscopy. These corpuscles are composed of 12 to 20 flattened Merkel cells and enclosed nerve terminals. The present study demonstrated for the first time argyrophilia in avian subepithelial Merkel cells with the use of Grimelius silver stain. Electron-microscopically, the Merkel cell was characterized by the presence of numerous densecore granules, approximately 80 to 140 nm in diameter, as well as specialized contacts with nerve terminals. The granules showed a tendency to accumulate in the cytoplasm in close association with both nerve terminals and basal lamina. This study also provided unequivocal evidence for exocytotic discharge of Merkel-cell granules at the plasma membrane facing not only the nerve terminals but also the basal lamina. The exocytotic figures toward the nerve terminals can be regarded as synaptic discharge of Merkel-cell granules, but the possibility also exists that the Merkel-cell granules may exert a trophic effect on the nerve terminals. The exocytotic release of Merkel-cell granules toward the basal lamina with no relation to nerve terminals may suggest an endocrine (paracrine) function for the Merkel cell. The avian subepithelial Merkel cells qualify as paraneurons, but their exact nature and function remain enigmatic as is the case of intraepithelial Merkel cells in other vertebrates.     

The fine structure of the eye of the mollusc Pecten maximus

Summary The retina of Pecten maximus is divided into two light sensitive layers forming the distal and proximal retinae. The cells from these layers have different electrophysiological responses, the distal cells giving primary off responses, and the proximal cells giving on responses. The receptor surfaces of the distal retinal cells are formed from lamellae produced by the outer membranes of flattened cilia. These cilia have a basal body, basal foot, no root system and a 9 + 0 internal filament content. Each cell gives rise to an axon from its distal side, and this process goes up to the basement membrane, which is present below the cellular lens, passes along beneath it, and joins the distal optic nerve. The receptor part of the proximal retinal cells is formed from a vast array of microvilli. Each of these cells also bears one or two cilia with a probable 9 + 0 internal filament complement and no roots. The proximal cells give rise to axons, forming the proximal optic nerve. Below the proximal retina is a reflecting layer, the argentea, and below this is a pigment cell layer.We would like to acknowledge the advice and encouragement of Professor A. F. Huxley, Professor J. Z. Young and Dr. E. G. Gray. — We would like to thank Mrs. J. I. Astafiev for drawing Fig. 1, Mr. S. Waterman for photographic help and Miss C. Martin for clerical assistance.     

Cooperativity of ganglioside-dependent with protein-dependent substratum adhesion and neurite extension of human neuroblastoma cells

The potential involvement of gangliosides in the adherence and neurite extension of human neuroblastoma cells (Platt and La-N1) was investigated on tissue culture substrata coated with the ganglioside GM1-binding protein, cholera toxin B (CTB) subunit, for comparison with similar processes on plasma fibronectin (pFN)-coated substrata. Cells attached with reduced efficiency on CTB substrata as compared with pFN substrata and required a much longer time to form neurite processes for a small percentage of cells on CTB. The specificity of these processes for GM1 binding was tested in a variety of ways. Supplementation of the cells with exogenous GM1, but not GD1a, identified a larger population of cells adherent on CTB (comparable to pFN-adherent cells) and dramatically increased the proportion of cells capable of forming neurites without reducing the time requirement. In ultrastructural studies using the scanning electron microscope (SEM) and immunofluorescence (IF) analyses to discriminate microtubule distributions, neurites of GM1-supplemented cells on CTB were virtually identical with pFN-adherent neurites, whereas unsupplemented cells on CTB generated processes with fine-structural differences. Treatment of cells during the GM1 supplementation period with cycloheximide completely abolished the ability of cells to generate neurites on CTB and decreased the adhesive capacity of cells as well; a similar treatment of cells had no adverse effect on adherence or neurite extension on pFN. The importance of one or more proteins in GM1-dependent processes was further confirmed by demonstrating the trypsin sensitivity of a cell surface component(s) required to achieve maximal attachment on CTB; in contrast, adherence and neurite extension on pFN were much more resistant to this treatment process. Therefore, these experiments demonstrate (a) that certain cell surface gangliosides are capable of mediating adherence and neurite outgrowth of human neuroblastoma cells on a suitable ganglioside-binding substratum; (b) this ganglioside dependence is cooperative with one or more cell surface proteins which can now be analysed. These results are discussed in light of the identification in ref. [16] (Exp cell res 169 (1987) 311) of a second ‘cell-binding’ domain on the pFN molecule competent for adherence and neurite extension of these neuroblastoma cells, as well as the potential role of pFN binding to a complex ganglioside on the surface of these neural tumor cells in these processes.     

Ultrastructure of excretory system in <Emphasis Type="Italic">Bothrioplana semperi</Emphasis> (Platyhelminthes,Turbellaria)

The ultrastructure of flame bulbs and epithelium of excretory canals in Bothrioplana semperi (Turbellaria, Seriata) have been studied. The flame bulbs consist of two cells, the terminal cell and the proximal canal cell. The weir is formed by two rows of longitudinal ribs. The ribs of the internal row originate from the flame cell, external ribs are formed by the proximal canal cell. Each external rib has a remarkable bundle of microfilaments, originating in the cytoplasm of the first canal cell distally to the bases of external ribs. Membrane of internal ribs is marked by small electrondense granules, separate or fused to an electron-dense layer, continuous to dense “membrane,” connecting both external and internal ribs. Sparse internal leptotrichs originate from the bottom of the flame bulb cavity. External leptotrichs are lacking. Septate junction is present only in proximal canal cell at the level of tips of cilia. The apical surface of the canal cell bears rare short microvilli. The basal membrane of canal cells forms long invaginations that may reach nearly the apical membrane. The epithelium of excretory canals lacks the cilia. The ultrastructure of flame bulbs and epithelium of the excretory canals in B. semperi shares representatives of suborder Proseriata (Seriata). The contradiction exists in interpretation of the structure of flame bulbs in Proseriata. Ehlers and Sopott-Ehlers assumed that the external ribs are derivatives of the proximal canal cell and internal ones are outgrowths of the terminal cell, while Rohde has found conversely: the external ribs are outgrowths of the terminal cell, the internal ones are outgrowths of the proximal canal cell. However, the illustrations provided by Rohde do not enable to ascertain what cells the internal and external ribs derive from, while illustrations provided by Ehlers justify his interpretation. The order of weir formation in B. semperi confirms the viewpoint of Ehlers. The implication of ultrastructure of flame bulbs in Proseriata, especially of the order of flame bulb formation, in the Platyhelminthes phylogeny has been discussed.     

Target cell contact suppresses neurite outgrowth from soma–soma paired Lymnaea neurons

Neurite extension from developing and/or regenerating neurons is terminated on contact with their specific synaptic partner cells. However, a direct relationship between the effects of target cell contact on neurite outgrowth suppression and synapse formation has not yet been demonstrated. To determine whether physical/synaptic contacts affect neurite extension from cultured cells, we utilized soma–soma synapses between the identified Lymnaea neurons. A presynaptic cell (right pedal dorsal 1, RPeD1) was paired either with its postsynaptic partner cells (visceral dorsal 4, VD4, and Visceral dorsal 2, VD2) or with a non‐target cell (visceral dorsal 1, VD1), and the interactions between their neurite outgrowth patterns and synapse formation were examined. Specifically, when cultured in brain conditioned medium (CM, contains growth‐promoting factors), RPeD1, VD4, and VD2 exhibited robust neurite outgrowth within 12–24 h of their isolation. Synapses, similar to those seen in vivo, developed between the neurites of these cells. RPeD1 did not, however, synapse with its non–target cell VD1, despite extensive neuritic overlap between the cells. When placed in a soma–soma configuration (somata juxtaposed against each other), appropriate synapses developed between the somata of RPeD1 and VD4 (inhibitory) and between RPeD1 and VD2 (excitatory). Interestingly, pairing RPeD1 with either of its synaptic partner (VD4 or VD2) resulted in a complete suppression of neurite outgrowth from both pre‐ and postsynaptic neurons, even though the cells were cultured in CM. A single cell in the same dish, however, extended elaborate neurites. Similarly, a postsynaptic cell (VD4) contact suppressed the rate of neurite extension from a previously sprouted RPeD1. This suppression of the presynaptic growth cone motility was also target cell contact specific. The neurite suppression from soma–soma paired cells was transient, and neuronal sprouting began after a delay of 48–72 h. In contrast, when paired with VD1, both RPeD1 and this non‐target cell exhibited robust neurite outgrowth. We demonstrate that this neurite suppression from soma–soma paired cells was target cell contact/synapse specific and Ca²⁺ dependent. Specifically, soma–soma pairing in CM containing either lower external Ca²⁺ concentration (50% of its control level) or Cd²⁺ resulted in robust neurite outgrowth from both cells; however, the incidence of synapse formation between the paired cells was significantly reduced. Taken together, our data show that contact (physical and/or synaptic) between synaptic partners strongly influence neurite outgrowth patterns of both pre‐ and postsynaptic neurons in a time‐dependent and cell‐specific manner. Moreover, our data also suggest that neurite outgrowth and synapse formation are differentially regulated by external Ca²⁺ concentration. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 357–369, 2000     

Ultrastructure of the taste disc in the red-bellied toad Bombina orientalis (Discoglossidae,Salientia)

The taste disc of the red-bellied toad Bombina orientalis (Discoglossidae) has been investigated by light and electron microscopy and compared with that of Rana pipiens (Ranidae). Unlike the frog, B. orientalis possesses a disc-shaped tongue that cannot be ejected for capture of prey. The taste discs are located on the top of fungiform papillae. They are smaller than those in Ranidae, and are not surrounded by a ring of ciliated cells. Ultrastructurally, five types of cells can be identified (mucus cells, wing cells, sensory cells, and both Merkel cell-like basal cells and undifferentiated basal cells). Mucus cells are the main secretory cells of the taste disc and occupy most of the surface area. Their basal processes do not synapse on nerve fibers. Wing cells have sheet-like apical processes and envelop the mucus cells. They contain lysosomes and multivesicular bodies. Two types of sensory cells reach the surface of the taste disc; apically, they are distinguished by either a brush-like arrangement of microvilli or a rod-like protrusion. They are invaginated into lateral folds of mucus cells and wing cells. In contrast to the situation in R. pipiens, sensory cells of B. orientalis do not contain dark secretory granules in the perinuclear region. Synaptic connections occur between sensory cells (presynaptic sites) and nerve fibers. Merkel cell-like basal cells do not synapse onto sensory cells, but synapse-like connections exist between Merkel cell-like basal cells (presynaptic site) and nerve fibers.     

Neuronal differentiation in cultures of weaver (wv) mutant mouse cerebellum

In the present study we report for the first time a weaver (wv) gene dose effect on neuron survival and neurite formation in vitro. Dissociated cerebellar cells from postnatal 7- and 8-day-old normal ( + / + ), heterozygous weaver ( + /wv) and homozygous weaver (wv/wv) mice were cultured as monolayers on poly-L-lysine coated glass. Cell death occurred rapidly in wv/wv cultures. Cell counts showed that less than 20% of the total neurons and neuronal precursors (identified by “birthday” radiolabeling techniques) survived by Day 3. Cell death was less extensive in + /wv cultures with 65% of the total neurons and 80% of the precursors surviving by Day 3. In contrast to wv/wv cultures, younger neurons survive better than the total population in + /wv cultures. The impairment of neurite formation over the first week is also proportional to the number of mutant genes as shown by quantitation of (a) the percentage of cells with neurites; (b) the percentage of cells with neurites of a given length class with time; (c) the lengths of the longest processes formed per cell. The mean longest neurite lengths obtained by computer digitization at 6 days in vitro were 41.8, 26.8, and 9.0 μm for + / +, + /wv, and wv/wv granule cells, respectively.     

Ultrastructure of the tubular nephron of the lizard Podarcis (= Lacerta) Taurica

The proximal, intermediate, and distal convoluted tubules of the neprhon of Podarcis (= Lacerta) taurica were examined by electron microscopy. Proximal tubule cells have large, apical cytoplasmic protrusions and microvilli interpreted to function in urate secretion. Adjacent cells are bound apically by tight junctions and desmosomes but interdigitate in their basal region. This situation is repeated in the other tubules with significant differences in intercellular space width. The basal surfaces bear numerous cytoplasmic processes. The intermediate tubule has proximal and distal segments each with dark, ciliated, and light cells, the cuboidal dark cells with dense cytoplasm constituting the main bulk of the wall. As the cells of the proximal and distal segments resemble those of the proximal and distal convoluted tubules, respectively, the intermediate tubule is considered as a transition region. The ciliated cell body has two broad processes extending from the lumen, one to the basement membrane and one to a foot process of a light cell. The light cell is surrounded by dark and ciliated cells. It does not reach the lumen, but contacts the basement membrane through a process running below a ciliated cell to form a mushroom-shaped structure in tubule cross-section, the light cell process forming the stalk and a ciliated cell the cap. The cilia probably propel the glomerular filtrate towards the distal convoluted tubule. This latter tubule has initial, middle, and terminal zones, all nonciliated but with different lumen widths and cell shapes.     

Arterial cells and CNS sheath cells from Aplysia californica produce factors that enhance neurite outgrowth in co-cultured neurons

Substrate-bound and soluble factors regulate neurite outgrowth and synapse formation during development, regeneration, and learning and memory. We report that sheath cells from CNS connectives and arterial cells from the anterior aorta of the sea slug, Aplysia californica, enhance neurite outgrowth from co-cultured Aplysia neurons. Sheath and arterial cell cultures contain several cell types, including fibrocytes, myocytes, and amoebocytes. When compared to controls (neurons with defined growth medium alone), the percentage of neurons with growth and the average neurite lengths are significantly enhanced by sheath and arterial cells at 48 h after plating of the neurons; these parameters are comparable to those of neurons cultured in medium containing hemolymph. Our results indicate that sheath cells produce substrate-bound factor(s) and arterial cells produce diffusible factor(s) that promote growth. These growth factors likely promote neuron survival and neurite outgrowth during neural plasticity exhibited in the adult CNS. Electronic Publication     

Possible glutaminergic interaction between the capsule and neurite of Pacinian corpuscles

The role of the capsule encasing the Pacinian corpuscle's (PC's) neurite, where mechanotransduction occurs, may be more than mechanical. The inner core of the PC's capsule consists of lamellar cells that are of Schwann-cell origin. Previously, we found both voltage-gated Na+ and K+ channels in these inner-core lamellae. Research on astrocytes and Schwann cells shows bidirectional signaling between glia and neurons, a major component of which is glutamate. Furthermore, Merkel cells show positive immunoreactivity for glutamate receptor mGluR5, and the glutamate-receptor antagonist kynurenate greatly decreases the static activity of the slowly adapting neurons of Merkel cell-neurite complexes. To investigate the possibility of glutaminergic interaction in PCs, we applied antibodies to glutamate, glutamate receptors, glutamate transporters, and SNARE proteins to cat mesenteric PC sections. Positive labeling was seen in the inner-core lamellae, at inter-lamellar connections, where the lamellae contact the membrane of the neurite and at the lamellar tips. The presence of these proteins on the lamellae and neurite membranes, demonstrated both with immunofluorescent light microscopy as well as immunogold electron microscopy, suggests a chemical, possibly bidirectional, interaction between the lamellar cells and the neurite. Thus, the capsule of the PC, apart from having a mechanical filtering function, may also provide an environment for lamellar-neurite interaction, perhaps acting as a neuro-modulator of the initiation, and/or continuation, of the mechanical-electrical transduction process. At the very least, the presence of the aforementioned proteins suggest some sort of "synaptic-like" activity in these mechanoreceptors, which up until now has not been considered possible.