Immunohistochemical demonstration of calbindin-containing nerve endings in the rat esophagus

Immunoreactivity for calbindin was found in nerve endings with irregular laminar shapes in the rat esophagus. In the myenteric ganglia, laminar endings of a range of sizes formed a complex network and appeared to lie at the surface of the ganglion. The myenteric ganglia that contained nerve endings were most abundant in the upper portion of the eosphagus, their number decreasing orally to anally. Calbindin-immunoreactive nerve cell bodies were scattered throughout the esophagus. Laminar terminals were found in the connective tissue of the lamina propria immediately beneath the epithelium and in the muscularis mucosae. Occasional nerve branches formed a network of aborizing endings that surrounded part of the submucosal arterioles. Immunoreactive nerve endings in the mucosa and submucosa were present only in the upper part of the cervical esophagus. Unilateral vagotomy caused a remarkable decrease in the number of the myenteric ganglia containing the calbindin-immunoreactive laminar endings after 15 days or survival; in some of ganglia, the laminar structures disappeared and nerve endings showing weak immunoreactivity had an indistinct appearance, so that the outline of the ganglia became obscure. In operated rats at 24 days, the number of innervated ganglia was about half that in normal rats. However, there was no change in the morphology and the occurrence of the immunoreactive laminar structures in the mucosa and submucosa after denervation. The results show that many of the laminar endings that are immunoreactive for calbindin in the myenteric ganglia are derived from the vagus nerve. Thus, the calbindin-immunoreactive nerve endings with laminar expansions that are found in the rat eosphageal wall could be sensory receptors.     

Target-specific innervation by autonomic and sensory nerve fibers in hairy fetal skin transplanted into the anterior eye chamber of adult rat

Summary Pieces of hairy skin tissue of fetal rat were transplanted into the anterior eye chamber of adult rats. The ability of autonomic and sensory nerve fibers from the host iris to innervate the grafted skin tissue was immunohistochemically and enzyme-histochemically examined using antisera against tyrosine hydroxylase (TH), substance P (SP), calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP), and a reaction medium for acetylcholinesterase (AchE). The grafted tissue was successfully implanted and connected with the host iris. Epidermis, dermis, subcutaneous tissue, hairs, hair follicles, sebaceous glands, and piloerector muscles developed in the graft. Two weeks after transplantation, TH-, SP-, and CGRP-immunoreactive fibers were observed in association with the blood vessels in the graft. Four weeks after transplantation, TH-immunoreactive fibers were distributed in the piloerector muscles, whereas SP-and CGRP-immunoreactive fibers were present around the hair follicles. VIP-immunoreactive and AchE-positive fibers were restricted to the host iris at all survival times. These results suggest that the outgrowth of autonomic and sensory nerve fibers from the host iris show target specificity for the grafted skin tissue.     

Time-related changes in the labeling pattern of motor and sensory neurons innervating the gastrocnemius muscle,as revealed by the retrograde transport of the cholera toxin B subunit

Summary Morphological changes in the motor and sensory neurons in the lumbar spinal cord and the dorsal root ganglia were investigated at different survival times following the injection of the B subunit of cholera toxin (CTB) into the medial gastrocnemius muscle. Unconjugated CTB, visualized immunohistochemically, was found to be retrogradely transported through ventral and dorsal roots to motor neurons in the anterior horn, each lamina in the posterior horn, and ganglion cells in the dorsal root ganglia at L₃–L₆. The largest numbers of labeled motor neurons and ganglion cells were observed 72 h after the injection of CTB. Thereafter, labeled ganglion cells were significantly decreased in number, whereas the amount of labeled motor neurons showed a slight reduction. Motor neurons had extensive dendritic trees filled with CTB, reaching lamina VII and even the pia mater of the lateral funiculus. Labeling was also seen in the posterior horn, but the central and medial parts of laminae II and III had the most extensively labeled varicose fibers, the origin of which was the dorsal root ganglion cells. The results indicate that CTB is taken up by nerve terminals and can serve as a sensitive retrogradely transported marker for identifying neurons that innervate a specific muscle.     

Filiform papillae as a sensory apparatus in the tongue: An immunohistochemical study of nervous elements by use of neurofilamcnt protein (NFP) and S-100 protein antibodies

Summary Nervous elements supplying the filiform papillae of the tongue of cattle and rats were investigated using immunohistochemistry against neurofilament protein (NFP) and glia-specific S-100 protein. The rod-shaped bovine filiform papillae were heavily keratinized along their entire length and lacked the connective tissue core that occurs in other mammals. Instead, the core was located posterior to the filiform papilla. The base of the bovine filiform papillae was invaded vertically by laminar connective tissue papillae. The core contained a large number of NFP-positive nerve fibers, most of them terminating as free endings in its anterior margin. NFP-positive nerves gathered around the anterior ridge of the epithelium at the base of the core and occasionally penetrated into the epithelium. The laminar connective tissue papillae at the base of the filiform papilla also contained NFP-positive nerve fibers. The core contained S-100-immunoreactive lamellated corpuscles, which were identified as simple corpuscles in electron micrographs. The structure and innervation of the bovine filiform papilla suggest that they represent a specialized sensory apparatus. The pyramidal filiform papillae of the rat were smaller, each containing a simple connective tissue core. Few NFP-positive nerve fibers from the nerve plexus entered the core. Filiform papillae are thus less specialized in rats than in cattle.     

Basic fibroblast growth factor-like immunoreactivity in the rat trigeminal sensory system and peri-oral skin with vibrissae

We have characterized an antiserum against basic fibroblast growth factor (bFGF) by immunoblot, investigated the location of bFGF-like immunoreactivity (bFGF-IR) in the trigeminal sensory system and perioral skin endowed with vibrissae, and demonstrated the site of bFGF mRNA expression in the vibrissae by in situ hybridization histochemistry. Light-microscopic immunohistochemistry has demonstrated that bFGF-IR is present not only in trigeminal ganglion neurons and their central and peripheral processes, but also in cells of the matrix, external root sheath and papillae of vibrissae and the stratum basale of the stratified squamous epithelium of the skin. Electron microscopy has revealed intense bFGF-IR mainly in cytoplasmic regions, other than the lumen of rough endoplasmic reticulum and the Golgi apparatus, in trigeminal ganglion neurons, in fibroblast-like cells in the papillae, and in capsules of vibrissae. In contrast, actively proliferating and/or differentiating cells in the matrix of vibrissae have intensely stained euchromatin and weakly labeled cytoplasm that, unlike that of the aforementioned cells, contain immunoreaction products in discrete spots less than 100 nm in diameter, implying the generation of different molecular forms of bFGF in cells of the matrix and papillae. Moreover, the accumulation of bFGF in the euchromatin appears to take place in cells at non-mitotic stages (possibly interphases), characterized by a conspicuous nucleolus and well-developed nuclear envelope. A digoxigenin-labeled cRNA probe for the demonstration of bFGF mRNA gives conspicuous hybridization signals mainly in the matrix of vibrissae. These findings suggest that bFGF is involved in the growth and differentiation of matrix cells during certain periods of the cell cycle and that it acts as a non-mitogenic mediator in the adult trigeminal sensory system.     

Glucagon-like immunoreactivity in hypothalamic neurons of the rat

Summary Antisera specific for three different regions of pancreatic proglucagon were used to examine the distribution of such immunoreactivity in rat hypothalamus. Neurons in the supraoptic and paraventricular nuclei were immunoreactive with an antiserum against glucagon, but not with antisera directed towards the aminoterminal region of proglucagon (glicentin) or the glucagon-like peptide I sequence in the carboxyl-terminal region of proglucagon. These findings confirm a previous report of glucagon-like immunoreactivity in the supraoptic and paraventricular nuclei, but indicate that, while this material is immunochemically related to glucagon, it is not derived from a proglucagon-like precursor.     

Postnatal development of autonomic and sensory innervation of thoracic hairy skin in the rat

Summary Histochemical, immunocytochemical, and radioenzymatic techniques were used to examine the neurotransmitter-related properties of the innervation of thoracic hairy skin in rats during adulthood and postnatal development. In the adult, catecholamine-containing fibers were associated with blood vessels and piloerector muscles, and ran in nerve bundles throughout the dermis. The distribution of tyrosine hydroxylase (TH)-immunoreactive (IR) fibers was identical. Neuronal fibers displaying neuropeptide Y (NPY) immunoreactivity were seen in association with blood vessels. Double-labeling studies suggested that most, if not all, NPY-IR fibers were also TH-IR and likewise most, if not all, vessel-associated TH-IR fibers were also NPY-IR. Calcitonin gene-related peptide (CGRP)-IR fibers were observed near and penetrating into the epidermis, in close association with hair follicles and blood vessels, and in nerve bundles. A similar distribution of substance P (SP)-IR fibers was evident. In adult animals treated as neonates with the sympathetic neurotoxin 6-hydroxydopamine, a virtual absence of TH-IR and NPY-IR fibers was observed, whereas the distribution of CGRP-IR and SP-IR fibers appeared unaltered. During postnatal development, a generalized increase in the number, fluorescence intensity, and varicose morphology of neuronal fibers displaying catecholamine fluorescence, NPY-IR, CGRP-IR, and SP-IR was observed. By postnatal day 21, the distribution of the above fibers had reached essentially adult levels, although the density of epidermal-associated CGRP-IR and SP-IR fibers was significantly greater than in the adult. The following were not evident in thoracic hairy skin at any timepoint examined: choline acetyltransferase activity, acetylcholinesterase histochemical staining or immunoreactivity, fibers displaying immunoreactivity to vasoactive intestinal peptide, cholecystokinin, or leucine-enkephalin. The present study demonstrates that the thoracic hairy skin in developing and adult rats receives an abundant sympathetic catecholaminergic and sensory innervation, but not a cholinergic innervation.     

Reticulum cells in the ontogeny of nasal-associated lymphoid tissue (NALT) in the rat

This study concerns the ontogeny of reticulum cells (RC) in the nasal-associated lymphoid tissue (NALT) of Wistar and Brown-Norway rats. A panel of monoclonal antibodies (mAb) directed against RC in peripheral lymphoid organs (antibodies ED10ED15) was used, together with a recently developed antibody ED17, which recognizes macrophages and Langerhans cells. Early in embryogenesis, staining with common connective tissue markers, ED14 and ED15, was found. ED17-positive cells were present before cells positive to ED1, a pan-macrophage marker, or Ia glycoproteins were observed. The first differentiation of reticulum was seen at the day of birth, when ED10 recognized a distinct area in the nasal mucosa. The first T-lymphocytes were found at the same time. Two days after birth, B-cells and ED11-positive cells were present in the NALT area. Fourteen days after birth, T- and B-cell compartments were recognizable. ED10 was found predominantly in the T-cell area and ED11 was mainly confined to the B-cell compartment. We conclude that the development of the NALT is closely accompanied by the phenotypic specialization of the reticulum. This suggests that the reticulum plays an important role in the compartmentalization of NALT tissue and in the retention of lymphocyte subsets within these compartments.     

Plasticity in developing rat uterine sensory nerves: the role of NGF and TrkA

In the present study we investigated the effects of infantile/prepubertal chronic oestrogen treatment, chemical sympathectomy with guanethidine and combined sympathectomy and chronic oestrogen treatment on developing sensory nerves of the rat uterus. Changes in sensory innervation were assessed quantitatively on uterine cryostat tissue sections stained for calcitonin gene-related peptide (CGRP). Uterine levels of NGF protein, using immunohistochemistry and ELISA, and mRNA, using Northern blots and in situ hybridization, were also measured. Finally, levels of TrkA NGF receptor in sensory neurons of T13 and L1 dorsal root ganglia (DRG), which supply the uterus, were assessed using densitometric immunohistochemistry. These studies showed that: (1) chronic oestrogen treatment led to an 83% reduction in the intercept density of CGRP-immunoreactive nerves; (2) sympathectomy had no effect on the density of uterine sensory nerves or on the pattern of oestrogen-induced changes; (3) NGF mRNA and protein increased following sympathectomy or chronic oestrogen treatment; and (4) oestrogen produced increased intensity of labelling (28%) for TrkA receptors in small-diameter sensory neurons, but decreased labelling (13%) in medium-sized neurons, which represent the large majority of the DRG neurons supplying the upper part of the uterine horn. Contrary to expectations, increased levels of NGF after sympathectomy and oestrogen treatment did not lead to increased sensory innervation of the uterus. The possibility that alterations in neuronal levels of TrkA contribute to the lack of response of uterine sensory nerves to the oestrogen-induced increase in NGF levels is discussed.This work was supported by The Wellcome Trust, UK (CRIG Grant 058122/Z/99/Z/JC/KO), and PEDECIBA, Universidad de la República, Montevideo, Uruguay     

Origin of the met-enkephalinergic innervation of the lateral septum in the rat

Summary The location of the cells giving rise to the methionine-enkephalin (Met-Enk)-ergic innervation of the lateral septal nucleus has been investigated in the rat by combining immunohistochemistry and retrograde axonal tracing. Small volumes (0.06 l) of apo-horseradish peroxidase (Apo-HRP) conjugated to wheat-germ agglutinin (WGA) and coupled with colloidal gold particles (WGA-ApoHRP-gold) were injected into the lateral septum. The retrogradely labeled cell bodies were visualized by silver intensification of the gold particles on Vibratome sections that were subsequently processed for immunohistochemistry for Met-Enk. Cells labeled with WGA-ApoHRP-gold were observed in the septal area, throughout the hypothalamus (mainly in the perifornical and lateral nuclei) and in the mesencephalon. The localization of Met-Enk-immunoreactive cells was as previously described. With the exception of a few septal cells close to the injection site, doubly labeled cells were found only in the perifornical nucleus of the hypothalamus. Almost all perifornical magnocellular cells were doubly labeled ipsilateral to the injection site, whereas on the opposite side, only about 25% of the Met-Enk-immunoreactive cells contained WGA-ApoHRP-gold. Other brain regions containing retrogradely labeled or Met-Enk-immunoreactive cells (particularly the raphe nuclei) did not show double-labeled neurons. This study demonstrates, using a new and sensitive technique for specific neurochemical tracing of tracts, that the origin of the Met-Enk-ergic innervation of the rat lateral septal nuclei lies in the magnocellular perifornical nuclei of the hypothalamus. The precise involvement of this pathway in limbic functions remains to be determined.     

Lymphoid and non-lymphoid cells in nasal-associated lymphoid tissue (NALT) in the rat

Summary Lymphocyte and macrophage subpopulations and the stroma of mucosa-associated lymphoid tissue in the nasal cavity of the rat were examined by application of immunohistochemical and enzyme histochemical methods to cryostat sections. Nasal-associated lymphoid tissue was composed of a loose reticular network with lymphocytes and macrophages, covered by epithelium. The epithelium was infiltrated with B cells, T helper (W3/13-positive) and T suppressor/cytotoxic or large granular cells (OX8-positive), ED1-positive macrophages and Ia-positive cells. The B cell areas were populated by B cells, immunopositive for surface IgM or IgG. B cells with surface IgA or IgE were rare. Germinal centres were found infrequently. T helper cells were scattered throughout the B cell area. A few ED1-positive macrophages and ED5-positive follicular dendritic cells were observed. Strong Ia staining (mostly of B cells) was found in this area. The T cell areas contained T helper and T suppressor/cytotoxic cells in about equal amounts, and numerous ED1-positive macrophages. ED1 staining was also found in the subepithelial area. Numerous ED1-, ED2- and ED3-positive macrophages were found in the border between the lymphoid mass and the surrounding connective tissue. A few non-lymphoid cells showed weak acid phosphatase or non-specific esterase activity. The morphological observations suggest that nasal-associated lymphoid tissue plays an important role in the first contact with inhaled antigens.     

Ultrastructure of free nerve endings in respiratory and squamous epithelium on the rat nasal septum

The distribution of nerve fibres in the mucosa of the nasal septum of the rat was investigated by means of transmission electron microscopy on transverse and tangential ultrathin sections. Near the basement membrane of respiratory and squamous epithelium, a rather dense network of unmyelinated nerve fibres occurs. Some fibres in the respiratory epithelium ascend between the epithelial cells to reach up to the tight junctions. These fibres appeared in transverse sections to end as hooks or boutons, sometimes with branches. These shapes resemble the free nerve endings that are considered to act as nociceptors. The small intraepithelial fibres, with diameters of about 0.5–1 m, contain both dense granules and clear vesicles comparable to synaptic vesicles. Substance P was found in dense granules in basal fibres; vasoactive intestinal peptide was absent throughout the epithelium. Acetylcholinesterase activity was observed closely associated with the basal fibres; the apical fibres showed little if any activity. Membrane specializations pointing to an efferent function as well as structures usually associated with mechanoreceptive functions were lacking in both respiratory and squamous epithelium.Part of this work was presented at the Annual Conference of the Netherlands Society for Electron Microscopy, 28–29 Nov 1985, Wageningen, The Netherlands. See Spit BJ and Hendriksen EGJ (1986) Ultramicroscopy 19:102–103     

Influence of a short light pulse at night on the ultrastructure of the rat pinealocyte: a quantitative study

Summary Although it is generally known that light strongly influences N-acetyltransferase activity and melatonin production in the pineal gland, little information is available concerning morphological changes following light exposure. As exposure of rats to a short light pulse at night rapidly depresses melatonin synthesis, we decided to determine whether this experimental condition produces rapid changes in the pinealocyte organelles. A 30-min light pulse at night (six hours after lights out) provoked rapid changes in the relative volumes of some pinealocyte organelles. The volume fractions of mitochondria, Golgi apparatus and lipid droplets, and the numbers of dense-core vesicles and synaptic ribbons decreased, whereas the volume fraction of lysosomes increased. There were no differences in the volumes of granular endoplasmic reticulum and vacuoles containing flocculent material in those animals exposed to light compared with control animals. These results indicate that a short light pulse at night causes ultrastructural changes that can be interpreted as morphological features of diminished activity of pinealocytes.     

Dynamics of the transganglionic movement of horseradish peroxidase in primary sensory neurons

Summary The dynamics of horseradish peroxidase (HRP) transport in primary sensory neurons were studied in rats by demonstration of the reaction product in spinal nerves, spinal ganglia, dorsal roots and in the spinal cord at different survival times after application of the enzyme to the transected sciatic nerve and to the spinal cord. Using tetramethylbenzidine as the chromogen according to Mesulam (1978), transganglionic transport of HRP was shown in both the disto-proximal direction after peripheral application, and proximo-distal direction after central application. Significant differences in staining intensity between the central and peripheral processes of primary sensory neurons were found after all survival times used in this study. After peripheral application the number of labeled axons and the staining intensity were higher in spinal nerves than in dorsal roots; an inverse situation occurred after central application. These differences as well as the time sequences in staining of different parts of primary sensory neurons suggest that HRP applied to a peripheral nerve and to the spinal cord, respectively, enters the perikarya of spinal ganglion cells in any case before continuing its movement in a cellulifugal direction. Lysosomal degradation of the major portion of the applied HRP is supposed. However, in the post-perikaryal portion of a considerable number of neurons HRP-transport still occurs to a varying extent, thus resulting in labeling of nerve endings. In some neurons a post-perikaryal transport could not be detected light microscopically. The transport rates differ: the calculated transport rate of disto-proximal, cellulipetal movement in the fastest transporting neurons was 7.5 mm/h, that of the disto-proximal cellulifugal movement 2.5 to 3 mm/h.This work was partly supported by the Hartmann Müller-Stiftung I want to thank Miss Regula Eichholzer for the technical assistance     

Distribution of prosaposin in the rat nervous system

Prosaposin is the precursor of four sphingolipid activator proteins (saposins A, B, C, and D) for lysosomal hydrolases and is abundant in the nervous system and muscle. In addition to its role as a precursor of saposins in lysosomes, intact prosaposin has neurotrophic effects in vivo or in vitro when supplied exogenously. We examined the distribution of prosaposin in the central and peripheral nervous systems and its intracellular distribution. Using a monospecific antisaposin D antibody that crossreacts with prosaposin but not with saposins A, B, or C, immunoblot experiments showed that both the central and peripheral nervous systems express unprocessed prosaposin and little saposin D. Using the antisaposin D antibodies, we demonstrated that prosaposin is abundant in almost all neurons of both the central and peripheral nervous systems, including autonomic nerves, as well as motor and sensory nerves. Immunoelectron microscopy using double staining with antisaposin D and anticathepsin D antibodies showed strong prosaposin immunoreactivity mainly in the lysosomal granules in the neurons in both the central and peripheral nervous systems. The expression of prosaposin mRNA, examined using in situ hybridization, was observed in these same neurons. Our results suggest that prosaposin is synthesized ubiquitously in neurons of both the central and peripheral nervous systems. Funding: This study was supported by the Ehime University INCS and in part by grants-in-aid for Scientific Research to S.M. (Exploratory Res. 19659380) from the Japan Society for the Promotion of Science and to AS (Priority Areas 18023029) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.     

Calbindin D-28k-positive neurons in the rat olfactory bulb

Summary We have studied the distribution of calbindin D-28k immunoreactivity in the rat olfactory bulb using specific monoclonal antibodies and the avidin-biotin-immunoperoxidase method. The largest number of positive neurons was located in the periglomerular layer. These neurons were identified as periglomerular cells; they have been described also by other authors as calbindin-positive elements. Close to these neurons, a second population of nerve cells was identified as superficial shortaxon neurons. The remaining layers showed a smaller number of stained elements. Other labeled neurons were located along the external border of the external plexiform layer; the scarce neurons marking its internal border were identified as van Gehuchten cells. No immunoreactive structures were found in the mitral cell layer, although we observed another population of immunostained short-axon cells at its internal border. Some reactive structures, identified by us as horizontal and vertical cells of Cajal, were located in the boundary zone between the internal plexiform layer and the granule layer. In the white matter, we found a neuronal type characterized by its large size and oriented arborization of varicose dendrites.     

Expression of fascin-1, an actin-bundling protein,in migrating hepatoblasts during rat liver development

Fascin-1 is an actin-bundling protein localized at the core actin bundles within microvillar projections and filopodial extensions in migrating cells. It is expressed at a low level in normal epithelial cells, but at a high level in tumor cells, indicating its importance in the invasion and motility of tumor cells. In addition, fascin-1 is expressed in human and murine embryos, occurring at high levels especially in developing nervous tissues. In this study, we have investigated the expression patterns of fascin-1 immunohistochemically during the early stages of rat hepatogenesis. A high expression of fascin-1 was detected in the liver bud and hepatoblasts at embryonic day (ED) 10.5, ED11.5, and ED12.5. Expression fell by ED13.5 and was not detectable at ED14.5. These observations demonstrate that the expression of fascin-1 is correlated with the migration activity of hepatoblasts during the early stages of liver development in rats. This study was supported in part by grant-in-aid for scientific research from the Japan Society for the Promotion of Science and by a grant from the Smoking Research Foundation, Japan.     

An immunohistochemical study on the postnatal development of rat nasal-associated lymphoid tissue (NALT)

Summary This study concerns the development of nasal-associated lymphoid tissue in the rat, using immuno- and enzyme-histochemical staining techniques on cryostat sections. Nasal-associated lymphoid tissue is present at birth as a small accumulation of mainly T lymphocytes and non-lymphoid cells; B cells are rare. Distinct areas of T and B cells appear at 10 days after birth; by that time high endothelial venules are also observed. Intra-epithelial lymphocytes are present, most of them being T-helper cells. ED1+ macrophages are seen throughout the tissue. The proportion of ED1+cells does not change during ontogeny. ED2+cells (tissue macrophages) are present predominantly at the border between the lymphoid tissue and the surrounding connective tissue, in all age-groups. ED3+mononuclear cells are scattered throughout the nasal-associated lymphoid tissue of young animals. Later on, the ED3+ cells migrate into the border-area between lymphoid and connective tissue. Ia+ non-lymphoid cells in the nasal lymphoid tissue increase in number during ontogeny. Only a few of them show acid phosphatase activity, indicating that the proportion of classical scavenger macrophages is low. Some of them may be antigen presenting (dendritic) cells. Ia+ dendritic cells also occur between the epithelial cells. Moreover, some epithelial cells express the Ia marker.     

Immunoreactive Tamm-Horsfall protein in the kidney and skin of the frog Rana temporaria

Summary Tamm-Horsfall protein (THP) is the main protein in normal human urine, and is found in the thick limb of the Loop of Henle in human kidney, and in other mammalian species. The skin of the frog, Rana temporaria, has similar physiological properties to this mammalian kidney tissue. In the present study, an immunohistological method involving an antibody to human THP was used to investigate the distribution of this distinctive protein in frog kidney and skin, and to compare its distribution with that found in the kidney tubules of rat and rabbit. THP-positive material was detected in the distal renal tubules and nephric duct of frogs, and was also located in the superificial epidermis of skin. It is suggested that its presence in amphibian skin is consistent with the hypothesis that THP is an important component of tissues that absorb sodium and chloride ions, but remain impermeable to water.