Serotonin-like immunoreactivity in Merkel cells and their afferent neurons in touch domes from the hairy skin of rats.код для вставкиСкачать
THE ANATOMICAL RECORD 232:112-120 (1992) Serotonin-Like lmmunoreactivity in Merkel Cells and Their Afferent Neurons in Touch Domes From the Hairy Skin of Rats KATHLEEN B. ENGLISH, ZU,O-ZHONG WANG, NAIDA STAYNER, LARRY J. STENSAAS, HERVE MARTIN, AND ROBERT P. TUCKETT Department of Physiology, University of Utah School of Medicine, Salt Lake City, Utah ABSTRACT Immunoreactivity to serotonin was observed in Merkel cells as well as the afferent type I nerves terminating upon them in touch domes excised from the belly skin of rats. Type I nerves were strongly immunoreactive and could be traced through the dermis of the domal papilla. Merkel cell immunoreactivity was sometimes seen in the entire cell, but was often localized in the Merkel cell cytoplasm adjacent to nerve terminals and may have been in the terminals themselves. Domes were fixed by immersion in 4% paraformaldehyde-lysine-sodiumm-periodate (PLP) fixative at 4°C for 2.5-3 hours and cryoprotected in 30% sucrose overnight. Sections were processed with the avidin-biotin complex peroxidase (ABC), peroxidase-antiperoxidase(PAP), and indirect immunofluorescence techniques with rabbit antiserum generated against serotonin. Merkel cells are situated in the basal epidermis of the most tactually sensitive regions of vertebrate skin. They are found in fish (Lane and Whitear, 1977), amphibians (Crowe and Whitear, 1978) and mammals, including humans (Merkel, 1875; Smith, 1970). Their presence in so many classes implies they offer a selective advantage in those animals possessing them (Simon and Hiller, 19891,but just what that role is has yet to be verified. In the hairy skin of rats, Merkel cells have a high density in the upper cylindrical cuff of the external root sheath of vibrissae. They are also in the basal epidermis of touch domes located on trunk skin or the whisker pad (Nurse et al., 1983; Nurse and Diamond, 1984). Following depilation, domes are visible as small elevations (170-314 pm in diameter; English, 1977; Leon and McComas, 1984) rising above the skin surface. On the trunk of adult rats, they are spaced about 3-5 mm apart and are associated with large tylotrich hairs (Straile, 1960). Almost all Merkel cells are innervated (Pasche et al., 1990). A type I afferent fiber forms a discoid shaped terminal on the dermal side of a Merkel cell’s surface (Iggo and Muir, 1969; English et al., 1980). Merkel cells characteristically contain dense cored granules -100 nm in diameter that are polarized next to the nerve terminals (Fig. 1).Sometimes granules fuse at synapse-like junctions (Chen et al., 1973). Mechanical depression of touch domes elicits a n irregular, slowly adapting discharge in the afferent type I fibers (Iggo and Muir, 1969). There is physiological evidence in support of two conflicting hypotheses: either Merkel cells transduce mechanical energy into nerve impulses (Horch e t al., 1974; Findlater et al., 1987), or type I nerve endings are the true receptors (Diamond et al., 1988; Gottschaldt and Vahle-Hinz, 1981). In addition, Merkel cells have been proposed to be the following: (1)paracrine regulators of the epidermis (Moll et al., 1986), (2) targets for growing nerves (Scott et al., 1981; Diamond, 0 1992 WILEY-LISS, INC. 1982; Yasargil et al., 1988), (3) neuromodulators of the nerve terminal’s mechanosensitivity, or (4) developmental “positional labelers” for the central nervous system (Diamond et al., 1988).For any of these functions, the contents of the dense cored granules and the stimuli causing their release are likely to be of central importance. Because serotonin has previously been shown to excite type I cutaneous afferent neurons in vivo (Beck and Handwerker, 1974), the present immunohistochemical study was conducted to determine if i t might be found in Merkel cell-axonal complexes. If present, serotonin’s role as a neurotransmitter in this system could be tested in a recently modified in vitro skin preparation (Reeh, 1986). MATERIALS AND METHODS Adult Sprague Dawley rats of both sexes were anesthetized with a n intramuscular injection of ketamine hydrochloride (100 mgiml) mixed with acepromazine (10 mg/ml) at 10 parts to 1, followed by 4% chloral hydrate administered subcutaneously according to body weight. Because of their large size, domes were removed from the belly after the fur was clipped and depilated (Nair). The tissue was (1) immersed into ice-cold, freshly prepared 4% paraformaldehyde-lysine-sodium-m-peroidate (PLP) fixative in 0.1 M phosphate-buffered saline (PBS; pH 7.4) for 2.5-3 hours; (2) rinsed in PBS for 10 minutes, followed by 15% sucrose in PBS for 1 hour; and (3) left in 30% sucrose in PBS overnight at 4°C. Individual domes were oriented on their side for sectioning, snap frozen in OCT, sectioned at 5-10 km, and Received March 22, 1991; accepted May 10, 1991. Address reprint requests to Kathleen B. English, Ph.D., Department of Physiology, University of Utah School of Medicine, 410 Chipeta Way, Research Park, Salt Lake City, UT 84108. SEROTONIN I N MERKEL CELLS AND AFFERENT NERVES 113 Fig. 1. Electron micrograph of a Merkel cell (MI and its associated type I nerve terminal (NT) with numerous mitochondria. The touch dome was excised from an adult rat. The Merkel cell has dense cored granules (g) in its cytoplasm. Spine-like processes (*) extend among neighboring keratinocytes of the epidermis. Merkel cells are above the epidermal basement lamina (bl) and form desmosomes (d) with the keratinocytes. Scale bar = 0.5 pm. mounted on chrome alum-subbed slides. The tissue was processed for immunocytochemical staining using conventional peroxidase-antiperoxidase PAP (Sternberger, 1986), avidin-biotin complex peroxidase ABC (Hsu et al., 1981), and indirect immunofluorescence techniques (Coons, 1958). Sections were stained with rabbit primary antiserum to serotonin [1:5,000 (NT102 [lyophilizedl; Eugene Tek Int.) or 1:200-300 (AB125; Chemicon)] overnight a t 4°C. Blocking serum was 10% normal goat serum in 0.1 M PBS saline. All antibodies were diluted in 0.1 M PBS saline plus 1% normal goat serum and 1%crystalline grade bovine serum albumin (Sigma, A7638). A 10minute wash in 3% H,02 quenched endogenous peroxidase activity in the skin. Biotin in sebaceous glands was bound up with a n avidin-biotin blocking step (Vector). For the ABC and PAP techniques, the secondary antisera were biotinylated goat anti-rabbit (1:400; Vector) or goat antiserum to rabbit (1:lOO).The PAP complex was prediluted (Dakopatts). The reaction products were visualized by the diaminobenzidine (DAB) reaction, which was closely monitored under a brightfield microscope. DAB was freshly filtered and prepared each run using 10 mg DAB (Sigma), 20 mls 0.1 M PBS, pH 7.4, and 4 ~1 30% HzOz (Sigma). The secondary antiserum for indirect immunofluorescence was goat anti-rabbit IgG-L-Rhodamine [(1:200-1:400) Boehringer Manheiml. The specificity of the serotonin antiserum was checked by preabsorbing serotonin antiserum at working dilution with 5-HT bound to agarose (Sigma, H7136) or with serotonin creatinine sulfate (Sigma). Other controls consisted of inclusion of 2 mg/ml of polyL-lysine (Sigma #P0879) in the antiserum diluent (Scopsi et al., 19861, omission of the primary or secondary antiserum, or substituting primary antiserum with normal, nonimmune rabbit serum (Eugene Tek Int.). Rat carotid body served a s positive control tissue. RESULTS Serotonin-like immunoreactivity was observed in Merkel cells, in afferent type I neurons with Merkel cells (Fig. 2) and in dermal mast cells. Immunoreactivity in Merkel cells was often strongest in their cytoplasm adjacent to nerve terminals or in the terminals themselves (Figs. 3A, 4A). The reaction time for immunostaining to occur was approximately 2-5 min. Not every Merkel cell in a given section was immunoreactive, but roughly 25% were. Type I neurons were strongly immunoreactive (Figs. 2B, 4B,C). Immunostaining was identical for both antisera tested (NT102 114 K.B. ENGLISH ET AL. Fig. 2. A Arrows point to Merkel cells immunoreactive to serotonin in the basal epidermis of a touch dome from a n adult rat. ABC immunoperoxidase technique, interference contrast microscopy. Scale bar = 10.0 pm. B: Arrows point to Merkel cells in the epidermis, and the open arrow points to the type I nerve in the dermis of a touch dome. Both are imrnunoreactive to serotonin. ABC irnmunoperoxidase technique, interference microscopy. Scale bar = 10.0 pm. Fig. 3. A Section of a touch dome with Merkel cells (arrows) and their nerve terminals immunofluorescently labeled against serotonin. Scale bar = 10.0 km. B: Control section of a touch dome where normal rabbit serum was substituted for the primary antiserum. Sections receiving preabsorbed antiserum were identical in appearance. Scale bar = 10.0 pm. 116 K.R. ENGLISH E T AL. Fig. 4. A Touch dome from an adult rat. Arrows point to type I nerve terminals immunofluorescently labeled for serotonin. Scale bar = 10.0 pm. B,C: Open arrows point to immunofluorescent type I nerves labeled for serotonin. The nerves rise in the dermis and terminate upon Merkel cells (arrow) in the basal epidermis of touch domes. Scale bar = 10.0 pm. SEROTONIN IN MERKEL CELLS AND AFFERENT NERVES Ilyophilizedl Eugene Tek Int. and AB125 Chemicon). Glomus cells of the carotid body were immunoreactive for serotonin (Gronblad et al., 1983) and thus substantiated the specificity of the antisera tested. Control sections incubated without primary or secondary antisera failed to demonstrate any immunoreactivity, as did sections incubated with normal rabbit serum or antiserum preabsorbed with bound serotonin (Fig. 3B). Preabsorption with serotonin creatinine sulfate required 10-1M concentrations for immunostaining to be eliminated and was not reliably reproducible (perhaps because the antiserum was generated against serotonin in its conjugated form). Inclusion of po1y-Llysine in the antiserum diluent did not abolish staining in the Merkel cells or the type I nerves, indicating the immunoreaction was not due to nonspecific binding of basic peptides. DISCUSSION In the present investigation, serotonin-like immunoreactivity was found in Merkel cells and their afferent nerves in touch domes excised from the hairy skin of rats. This supports previous immunohistological studies demonstrating serotonin in Merkel cells from the eel (Zaccone, 1986) and pig (Garcia-Caballero et al., 1989b), as well as studies employing permanganate staining and reserpine treatment to demonstrate monoamines in Merkel cell granules of mice (Chao and Liang, 1975). However, it is a t variance with other less sensitive and selective histochemical and pharmacological investigations in cats and rats (Smith and Creech, 1967; Iggo and Muir, 1969; Hartschuh and Grube, 1979; see also Diamond et al., 1986). In rats, Merkel cells have been shown to contain the opioid peptide metenkephalin (Hartschuh e t al., 1979) and the putative neurotransmitter adenosine triphosphate (ATP; Crowe and Whitear, 1978; Nurse et al., 1983). In various other species, Merkel cells have been demonstrated to be immunoreactive for neuron specific enolase, chromogranin A, vasoactive intestinal polypeptide (VIP), substance P, pancreastatin, and calcitonin gene-related peptide (Alvarez et al., 1988; Gu et al., 1981; Hartschuh and Weihe, 1988, 1989). It is common for biogenic amines to be colocalized with transmitters and neuropeptides in excitatory or receptosecretory cells (Gauweiler et al., 1988; Kanagawa et al., 1986; Forloni et al., 1987). Indeed by definition, cells of the APUD (amine precursor uptake and decarboxylation), DNES (diffuse neuroendocrine system), or paraneuron class characteristically possess amines and peptides as well a s marker molecules such as neuron specific enolase and chromogranin A (Fujita, 1983; Pearse, 1986; Hartschuh and Weihe, 1988). In general, Merkel cells of the skin, along with chromaffin cells of the adrenal medulla, neuroepithelial cells of the lung, enterochromaffin cells of the gastrointestinal tract, and glomus cells of the carotid body are all considered to be neuroendocrine cells, belonging to the APUD, DNEs, paraneuron, or receptosecretory class of cells. The presence of biogenic amines in Merkel cells might therefore be expected, but it is not a n absolute requirement for the receptosecretory classification to stand. The present study expands the number of species in which serotonin has been found in Merkel cells. As 117 methodologies are refined, there may be more correlation between immunohistochemical profiles of Merkel cells (see Table 1).For instance, met-enkephalin was originally reported to only occur in Merkel cells from rodents (guinea pigs, mice, and hamsters), but not in those from cats, dogs, pigs, and humans, which demonstrated immunoreactivity to vasoactive intestinal polypeptide (VIP) instead (Hartschuh et al., 1984). Yet a t the same time other investigators found met-enkephalin immunoreactivity in Merkel cells from primates (Warner e t al., 1983). Unfortunately, there are technological difficulties with immunohistochemical staining. This is exemplified by synaptophysin reactivity only occurring in perfusion-fixed guinea pig tissue and not in immersion-fixed pig and human skin. Similarly, chromogranin A is shown with bovine-generated antiserum rather than porcine-generated antiserum (Hartschuh and Weihe, 1988). The existence of serotonin-like immunoreactivity both in Merkel cells and their afferent nerves in the present study was unexpected. It was found in Merkel cells but not afferent nerve fibers in the pig and eel (Zaccone, 1986; Garcia-Caballero et al., 198913). However, similar observations have been made in other APUD cells and their sensory nerve supply. For example, in the adrenal medulla, both adrenaline cells and their afferent neurons express immunoreactivity for enkephalins (Schultzberg et al., 1978; Pelto-Huikko et al., 1985). Similarly, catecholaminergic primary sensory neurons terminate upon catecholamine-containing type I glomus cells in the carotid body (Katz and Black, 1986). The physiological significance of dual localization of putative neurotransmitter substances in APUD cells and their afferent neurons is presently unknown. Earlier enzymatic histochemical studies of the type I nerve endings of Merkel cells indicated acetylcholinesterase and leucine aminopeptidase were present in rabbits, and alkaline phosphatase in cats and guinea pigs (Winkelmann, 1977). The present investigation is the first study showing a specific immunohistochemical reaction in the type I nerve terminals on Merkel cells. Other investigators have found CGRP immunoreactive nerve fibers occasionally associated with Merkel cells in the footpad of rats, but these were not the type I fibers of the Merkel cell-axonal complex (IshidaYamamoto et al., 1989). Serotonin is known to be ultrastructurally associated with synaptic vesicles (Sanders-Bush and Martin, 1982). Merkel cells have numerous dense cored granules, but their afferent neurons do not. There is, however, considerable experimental evidence that a large proportion of serotonin is stored in a cytoplasmic extravesicular form (Halaris and Freedman, 1977; Lewis and Moertel, 1978; Sanders-Bush and Martin, 1982). Therefore, the sparcity of dense-cored granules in the afferent nerves terminating upon Merkel cells does not exclude the possibility of serotonin being present in cytoplasmic stores. The role that serotonin plays in the Merkel cell-axonal complex remains to be determined. It may be involved in any one of several biological processes. For example, serotonin may be important in the generation of type I responses in afferent nerves because both serotonin formation and type I response are highly de- 118 K.B. ENGLISH ET AL. TABLE 1. Merkel cell immunohistochemistry Substance Met-enkephalin Substance P CGRP (calcitonin gene-related peptide) VIP PHYPMI (peptide histidine isoleucine) Pancreastatin Serotonin Bombesin NSE (neuron specific enolase) Cytokeratin 8,18 PGP (protein gene product 9.5) Synaptophysin Species rat guinea pig, mouse, hamster monkey adult and fetal pig adult cat, adult mouse, adult pig adult and fetal pig adult pig, adult cat, adult mouse adult cat adult pig dog, cat, human, pig adult cat, mouse, pig adult pig adult cat, mouse, pig adult and fetal pig adult human and pig eel Plg fetal human eel cat, rat adult and fetal human adult human rabbit, pig, human human human Pig human, rabbit, pig adult guinea pig adult and fetal pig adult cat, adult pig adult and fetal pig Chromogranin A s-100 pendent upon POz. Type I receptors fail to respond to mechanical stimulation in a n oxygen-depleted environment. Furthermore, Merkel cells become degranulated when touch domes are mechanically stimulated under hypoxic conditions (Findlater et al., 1987). On the other hand, serotonin may be required for synthesis and storage of polypeptides (Owman e t al., 1973). Alternatively, recent research indicates bidirectional communication exists between neuroendocrine cells and the immune system, wherein serotonergic systems inhibit immune responses (Warren e t al., 1990; Devoino et al., 1990). Now that serotonin has been identified in the Merkel cell-axon complex, its function will be the focus of future electrophysiological and neuropharmacoligical investigations. ACKNOWLEDGMENTS The authors wish to express thanks to Dr. Andy Towles of ETI, Dr. P. Burgess for his critique of the manusmirk. and to Vicki Skelton for twine: the manuscript. Supported by NIH grants" -NS%7938 and NS26229. LITERATURE CITED Alvarez, F.J., C. Cervantes, R. Villalba, I. Blasco, R. MartinezMurillo, J.M. Polak, and J. 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