Co-localization of vasoactive intestinal polypeptide and neuropeptide Y immunoreactivities in the nerve fibers of the rat adrenal gland

The co-localization of Vasoactive Intestinal Polypeptide (VIP) with Neuropeptide Y (NPY) or its C-flanking peptide (C-PON) was investigated with immunocytochemistry methods in the adrenal gland of the rat. Most of the VIP immunoreactive (+) nerve fibers found in the capsule/glomerular zone also exhibited NPY or C-PON immunoreactivity (IR). We found that at least two populations of VIP varicose nerve fibers can be observed, the most prevalent exhibited both VIP/NPY or VIP/C-PON IR and the other which was rather scarce lacked NPY or C-PON IR. In the superficial cortex VIP/NPY or VIP/C-PON IR nerve fibers were often associated with capsular or subcapsular vascularization and extended into the zona glomerulosa. In the deeper layers of the adrenal cortex radial fibers were closely associated with the inner vascularization of the zona fasciculata and reticularis. In the adrenal medulla NPY or C-PON immunoreactivity was associated with ganglion neurons as well as chromaffin cells; these last cells were always VIP (-). VIP and NPY/C-PON IR could be co-localized in catecholaminergic nerve terminals of the adrenal cortex but not in the adrenal medulla.     

Alterations in nociception following adrenal medullary transplants into the rat periaqueductal gray

Summary Adrenal medullary chromaffin cells were transplanted to the midbrain periaqueductal gray, a region known to play a primary role in the modulation of nociception. Chromaffin cells were chosen for transplantation since they contain several neuroactive substances (e.g. catecholamines, opioid peptides, other neuropeptides) whose release can be stimulated by pharmacological agents such as nicotine. Both dissected adult rat adrenal medullary tissue and bovine chromaffin cells served as graft tissue in the periaqueductal gray region of adult rats. When stimulated with a low dose of nicotine, potent analgesia was induced in the these animals as assessed by tail flick, paw pinch and hot plate tests. The bovine chromaffin cell implants were more effective in inducing this response. The analgesia induced by nicotine stimulation in rats with adrenal medullary implants was partially attenuated by both opiate antagonist naloxone, and adrenergic antagonist phentolamine. These results suggest that the implantation of cells which release neuroactive substances can produce reductions in pain sensitivity.     

Differential distribution of neuropeptides and serotonin in pig adrenal glands

A differential distribution of vasoactive neuropeptides and serotonin in chromaffin cells and nerve fibers within the adrenal glands of the pig (Sus scrofa) was found using immunohistochemical methods. Met- and leu-enkephalins, present at high levels in the medulla (measured by radioimmunoassay), occurred in adrenaline storing cells, some of which contained calcitonin gene-related peptide. Islets of chromaffin cells beneath the capsule also contained enkephalins and calcitonin gene-related peptide. Nerve fibers with enkephalin-like immunoreactivity were sparse, but many varicose fibers in the inner cortex and medulla showed calcitonin gene-related peptide immunofluorescence in a pattern similar to vasoactive intestinal polypeptide. Neuropeptide Y was mainly associated with perivascular fibers and neither neuropeptide Y nor vasoactive intestinal polypeptide immunoreactive chromaffin cells were detected. In contrast to the neuropeptides, most serotonin-like immunoreactivity coincided with noradrenaline histofluorescence. It is concluded that the distribution of nerve fibers with calcitonin gene-related peptide and vasoactive intestinal polypeptide would allow interactions between chromaffin and inner cortical cells. Stimuli activating noradrenaline chromaffin cells could release serotonin while stimulation of adrenaline storage cells would release enkephalin and, to a lesser extent, calcitonin gene-related peptide. Met-enkephalin, which occurs 3 4:1 over leu-enkephalin, is the most likely of the co-released peptides to reach distant receptors via the venous outflow.     

The distribution of immunoreactive chromogranins, S-100 protein, and vasoactive intestinal peptide in compound tumors of the adrenal medulla

Three adrenal medullary tumors that showed admixtures of pheochromocytomatous elements with ganglioneuroma or ganglioneuroblastoma were studied to determine the distribution of immunoreactive chromogranins, S-100 protein, and vasoactive intestinal peptide (VIP). Two tumors consisted of typical pheochromocytoma cells admixed with mature-appearing ganglioneuroma. The third consisted of pheochromocytoma admixed with ganglioneuroblastoma and contained many immature and cytologically atypical cells. In all cases, chromogranin staining was absent or weak in neuronal perikarya and moderate to intense in varicosities of neuronal processes, a finding consistent with the presumed distribution of secretory granules in neurons. Chromogranin staining was also intense in chromaffin cells. Glial cells that stained for S-100 were randomly scattered among chromaffin cells but accumulated in areas with neuronal processes. Weak staining for VIP was present in neuronal cells in one of the two tumors with ganglioneuromatous features. Intense staining for VIP occurred in the third tumor in both neuronal and apparently nonneuronal cells. We conclude that granule distribution and cell-cell interactions for specific cell types in compound tumors tend to mimic those in normal adrenal medulla and sympathetic ganglia. Although immunoreactive VIP was localized exclusively to neurons in one tumor, as in normal tissues, patterns of staining for VIP across tumors are less predictable.     

Localization and release of immunoreactive vasoactive intestinal polypeptide in bovine adrenal medulla

The concentration of immunoreactive (IR) vasoactive intestinal polypeptide (VIP) in extracts from bovine adrenal medulla was 29.9 +/- 7.2 pmol/g wet wt., which was about 100 times that of IR neurotensin and 30 times that of IR somatostatin. Chromatographic analysis showed that most of the IR-VIP was the same molecular size as synthetic VIP(1-28). On retrograde perfusion of isolated bovine adrenal gland, release of VIP with catecholamine (CA) was marked on stimulation with high K+, but slight on stimulation with acetylcholine, which induced marked release of CA. These results suggest that most of the VIP is localized not in CA storing granules in chromaffin cells, but in other intraadrenal neuronal components. In immunohistochemical studies, IR VIP fibers with large varicosities were observed around the vessels in the adrenal medulla.     

Immunological studies on the distribution of chromogranin A and B in endocrine and nervous tissues

Bovine chromaffin granules contain two major families of acidic proteins, chromogranins A and B. The occurrence of these proteins in endocrine and nervous tissue was investigated by immunoblotting (one- and two-dimensional), and by immunohistochemistry. Immunoblotting revealed that in anterior hypophysis and in splenic nerve from ox, immunologically crossreacting proteins are present which in two-dimensional electrophoresis migrate to the same position as adrenal chromogranins A and B. Smaller proteins derived from chromogranin B by endogenous proteolysis were much less prominent in these tissues when compared with adrenal medulla. Immunohistochemistry performed in rat and bovine tissues established that chromogranin B is present in all cells of the adrenal medulla. It is also found in the anterior hypophysis, the endocrine pancreas, in enterochromaffin and in sympathetic ganglion cells, but e.g. is absent from posterior hypophysis and exocrine tissues. It is concluded that chromogranins A and B have a widespread distribution in endocrine and nervous tissue. Proteolytic processing of chromogranin B in the storage organelles of hypophysis and splenic nerves is apparently slower than that in chromaffin granules. The widespread distribution of the chromogranins resembling that of neuropeptides is a clear indication for some special, yet to be discovered, function.     

Vasoactive intestinal peptide increases tyrosine hydroxylase activity in normal and neoplastic rat chromaffin cell cultures

Vasoactive intestinal peptide (VIP) acutely increases tyrosine hydroxylase (TH) activity in cultures of dispersed normal adult rat chromaffin cells and of PC12 rat pheochromocytoma cells. High concentrations of VIP (10 microM) produce about 3-fold increases in TH activity in both cell types. VIP also increases the content of cyclic adenosine 3':5'-monophosphate (cAMP) in PC12 cells. VIP may increase TH activity by promoting the cAMP-dependent phosphorylation of the enzyme. Nerve fibers containing VIP-like immunoreactive material have been reported in the adrenal medulla and in other catecholamine (CA)- storing tissues. This VIPergic innervation may regulate CA synthesis and other cAMP-dependent processes in these tissues.     

Synaptic activation of rat adrenal medulla examined with a large photodiode array in combination with a voltage-sensitive dye.

The adrenal medulla is innervated by sympathetic preganglionic nerve fibers in the splanchnic nerve. Synaptic activation of the adrenal medulla causes catecholamine secretion which is known to be modified by various neuropeptides and other factors. To understand the neuronal control mechanism of catecholamine secretion, it is necessary to know the transfer function at the synapse and how it is affected by such factors. By using a large photodiode array in combination with a voltage-sensitive dye, membrane potential changes in a slice of the rat adrenal gland were recorded upon brief local electrical stimulation. Electrical signals were recorded only on the portion of the diode array corresponding to the medulla. In a typical record, a spike and an underlying slow potential were observed following a small deflection due to a presynaptic nerve action potential. Both the spike and slow potential were blocked in Ca(2+)-free solution or by hexamethonium, a nicotinic antagonist, but were not affected by atropine, a muscarinic antagonist. The slow potential was interpreted as a nicotinic synaptic potential in the chromaffin cells and the spike as a population action potential. A double pulse experiment revealed that the chromaffin cell action potential began to fail only when the stimulus interval was less than 50 ms (20 Hz). When the stimulus intensity was reduced, the minimal response was found to behave in an all-or-none fashion. This suggested that one nerve fiber is innervating a cluster of chromaffin cells, which may correspond to a previously histologically identified "complex" of cells [Hillarp (1946) Acta. anat. 4, Suppl. 1]. Each complex was innervated by approximately four nerve fibers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulatory effect of vasoactive intestinal polypeptide on catecholamine secretion from isolated guinea pig adrenal chromaffin cells

The stimulatory effect of vasoactive intestinal polypeptide (VIP) on catecholamine (CA) secretion from isolated guinea pig adrenal chromaffin cell was studied. VIP (1-10 microM) induced dose-dependent CA secretion, which was slow and continued for at least 30 min. This VIP-induced CA secretion was dependent on the presence of Ca2+ in the medium, but no significant increase in Ca2+ uptake by the cells was observed during their stimulation with VIP. Studies on the intracellular free Ca2+ level ([Ca2+]i) using fura-2 showed that acetylcholine and muscarine induced a marked increase in the [Ca2+]i, but that VIP induced only a slight increase. Thus VIP may induce CA secretion by increasing the sensitivity of the secretion of CA to Ca2+.     

Possible autocrine regulation of chromaffin cell activity in adrenal glands of the frog by endothelin-1-induced serotonin release

The aim of the present study was the demonstration of mechanisms of regulation of activity of chromaffin cells in the adrenal gland of Rana ridibunda (Anura-Amphibia). Previous studies have shown that endothelin-1 is an important factor for the maintenance of adrenal gland function. On the basis of these findings, frogs were injected with [Ala(1,3,11,15)]-endothelin-1 (0.025 mg/0.2 ml), which is a selective agonist of the endothelin B receptor, whereas control animals were injected with Ringer solution (0.2 ml). The adrenal glands were removed at 5, 20, and 60 min after injection and fixed, embedded in paraffin wax and stained by histological and immunohistochemical means, applied on adjacent 4-microm-thick sections. Sections were stained with hematoxylin and eosin for overall tissue analysis and, in parallel, serotonin was localized using the streptavidin-biotin complex technique while dopamine beta-hydroxylase and serotonin 2A receptors were shown by the peroxidase-antiperoxidase (PAP)-3,3'-diaminobenzidine tetrachloride (DAB) method. After injection of [Ala(1,3,11,15)]-endothelin-1, chromaffin cells secreted serotonin and synthesized dopamine beta-hydroxylase. In conclusion, these findings suggest that [Ala(1,3,11,15)]-endothelin-1 stimulates chromaffin cell activity in frog adrenal glands. Moreover, the presence of serotonin 2A receptors in chromaffin cells indicates that these cells are also targets for serotonin and that there is an autocrine signaling pathway in chromaffin cells. This is the first report providing data on the effects of endothelin-1 on chromaffin cells in frog adrenal glands.     

Localization of angiotensin II binding sites in the bovine adrenal medulla using a labelled specific antagonist

Angiotensin II binding sites have been localized in sections of bovine adrenal glands and on living cultured bovine adrenal medullary cells using [125I]-[Sar1,Ile8]-angiotensin II and autoradiographic techniques. Binding sites were observed over both adrenaline and noradrenaline chromaffin cells. However, they were present in higher density over adrenaline cells, as determined by the distribution of phenylethanolamine N-methyltransferase mRNA by in situ hybridization histochemistry and of glyoxylic acid-induced fluorescence of noradrenaline. Binding sites were also observed in low density over nerve tracts within the bovine adrenal gland. Living cultured bovine adrenal medullary cells possessed angiotensin II binding sites. Not all cells were labelled. At least 73% of identified dispersed chromaffin cells in these cultures were labelled. Some chromaffin cells were not labelled with the ligand, and at least some non-chromaffin cells in the cultures did possess angiotensin II binding sites. The results provide direct anatomical support for the known ability of angiotensin II to elicit catecholamine secretion from perfused adrenal glands and from cultured adrenal chromaffin cells. They also suggest that some of the effects of angiotensin II on calcium fluxes and second messenger levels measured in cultured adrenal medullary cell preparations may be due to angiotensin II acting on non-chromaffin cells present in these cultures.     

Cografts of adrenal medulla with C6 glioma cells in rats with 6-hydroxydopamine-induced lesions

Amitotic [3H]thymidine-labeled C6 glioma cells, which are known to produce neurotrophic factor(s), were grafted alone and with adrenal chromaffin cells in an attempt to improve chromaffin cell survival and phenotypic differentiation. Long-Evans rats with unilateral 6-hydroxydopamine-induced lesions of the nigrostriatal pathway were divided into four groups: (1) those receiving adrenal medullary cells co-transplanted with C6 glioma cells; (2) those receiving adrenal medullary graft alone; (3) those receiving C6 glioma grafts alone; and (4) those serving as a vehicle control group. All rats were killed one month after transplantation. Immunohistochemical, neurochemical, and autoradiographic methods were used to identify and characterize the grafted cells. Tyrosine hydroxylase-immunoreactive cells were found in all animals that received grafts of the adrenal medulla alone or of adrenal medulla co-transplanted with C6 glioma cells. The cograft recipients had more tyrosine hydroxylase-immunoreactive cells than the hosts receiving just adrenal chromaffin cells (P less than 0.05). Additionally, more grafted chromaffin cells formed processes in the former group. All three tissue recipient groups (adrenal medullary, C6 glioma cell, and cografted animals) had a significant reduction (P less than 0.05) in ipsilateral rotations after amphetamine (0.5 mg/kg i.p.) injections as compared to the control vehicle recipient group. Moreover, the reduction in rotation was more marked in the cografted hosts than in the other two implanted groups (P less than 0.05). Significantly higher dopamine levels were found in the transplant sites of both cograft and adrenal medullary graft recipients than in sham grafted control animals.     

Ultrastructural evidence for the development of adrenal medullary grafts in the brain

Summary This study shows that mouse mature chromaffin cells can elaborate neurite-like fibers and became integrated with the host brain. A piece of adrenal medulla, with or without attached adrenal cortical tissue, was implanted into the subarachnoid space or the hippocampal formation and examined using the electron microscopy. One week after transplantation, chromaffin cells could be observed surrounded by a basal lamina, containing many densecored vesicles 100–280 nm in diameter, including synaptic-like vesicles, which tended to gather in the cytoplasmic area or processes. The cells were irregularly shaped and bore cytoplasmic processes which sometimes ended with thick growth cone-like structures. The Golgi complex seemed to be well developed, suggesting the synthesis of new storage vesicles. One month after transplantation, the vast majority of chromaffin cells showed the noradrenaline phenotype typical of noradrenaline-storing cells in the normal gland, irrespective of graft components used or implantation sites. Some cells, presumably corresponding to the adrenaline phenotype, had secretory vesicles (140–210 nm in diameter) with denser cores than in the adrenaline-storing cells of normal gland. In the subarachnoid space, both types of graft had mostly cuboid chromaffin cells which bore a few, short, blung cytoplasmic processes. In the intracerebral transplants, the chromaffin cells of cortex-free adrenal medullary grafts developed processes having the characteristics of neurites extending from the chromaffin cells, in contrast to their counterparts with attached adrenocortical tissue. Thin sections through both types of graft showed isolated nerve cells, morphologically similar to sympathetic neurons, in the neighbourhood of the chromaffin cells. Reinnervation of the chromaffin cells was frequently observed in cortex-free implants. The integration of these grafts in the host brain is strongly suggested.     

Re-establishment of neurochemical coding of preganglionic neurons innervating transplanted targets

We investigated the effect on neurochemical phenotype of changing the targets innervated by sympathetic preganglionic neurons. In neonatal rats, the adrenal gland was transplanted into the neck, to replace the postganglionic neurons of the superior cervical ganglion. Transplanted adrenal glands survived, and contained noradrenergic and adrenergic chromaffin cells, and adrenal ganglion cells. Retrograde tracing from the transplants showed that they were innervated by preganglionic neurons that would normally have supplied postganglionic neurons of the superior cervical ganglion. The neurochemical phenotypes of preganglionic axons innervating transplanted chromaffin cells were compared with those innervating the normal adrenal medulla or superior cervical ganglion neurons. As in the normal adrenal gland, preganglionic nerve fibres apposing transplanted chromaffin cells were cholinergic. The peptide and calcium-binding protein content of preganglionic fibres was similar in normal and transplanted adrenal glands. In both cases, cholinergic fibres immunoreactive for enkephalin targeted adrenergic chromaffin cells, whilst cholinergic fibres with co-localised calretinin-immunoreactivity innervated noradrenergic chromaffin cells and adrenal ganglion cells. In contrast to the innervation of normal adrenal glands, these axons lacked immunoreactivity to nitric oxide synthase. In a set of control experiments, the superior cervical ganglion was subjected to preganglionic denervation in rat pups the same age as those that received adrenal transplants, and the ganglion was allowed to be re-innervated over the same time course as the adrenal transplants were studied. When the superior cervical ganglion was re-innervated by preganglionic nerve fibres, we observed that all aspects of chemical coding were restored, including cholinergic markers, nitric oxide synthase, enkephalin, calcitonin gene-related peptide and calcium binding proteins in predicted combinations, although the density of nerve fibres was always lower in re-innervated ganglia. These data show that the neurochemical phenotypes expressed by preganglionic neurons re-innervating adrenal chromaffin cells are selective and similar to those seen in the normal adrenal gland. Two explanations are advanced: either that contact of preganglionic axons with novel target cells has induced a switch in their neurochemical phenotypes, or that there has been target-selective reinnervation by pre-existing fibres of appropriate phenotype. Regardless of which of these alternatives is correct, the restoration of normal preganglionic codes to the superior cervical ganglion following denervation supports the idea that the target tissue influences the neurochemistry of innervating preganglionic neurons.     

Possible role of insulin-like growth factor-II C-peptide on catecholamine release and ultrastructural aspects of chromaffin cells in the adrenal gland of the frog

The present study was undertaken to demonstrate that insulin-like growth factor-II C-peptide (IGF-II C-peptide) affects the function of the adrenal gland of Rana ridibunda (Anura, Amphibia) by stimulating chromaffin cells. Previous studies have shown that insulin-like growth factors affect adrenal gland function in mammals. On the basis of these findings, frogs were injected with IGF-II C-peptide (2.5 microg/0.2 ml), whereas control animals were injected with Ringer solution (0.2 ml). The adrenal glands were removed at 12 and 48 h after injection and fixed, embedded in paraffin wax and Epon, and examined by immunohistochemistry and transmission electron microscopy to investigate whether there were structural changes and activation of chromaffin cells in the frog adrenal gland. Sections were stained with hematoxylin and eosin for overall tissue analysis and, in parallel, serotonin was localized using the streptavidin-biotin complex technique while dopamine beta-hydroxylase was shown by the peroxidase-antiperoxidase-3, 3'-diaminobenzidine tetrachloride method. After injection of IGF-II C-peptide, chromaffin cells released serotonin and synthesized dopamine beta-hydroxylase. The most pronounced effect of IGF-II C-peptide on the chromaffin cells was observed at 12h after injection. Our results indicate that there is a possible role of IGF-II C-peptide on chromaffin cell activity enhancing catecholamine release in the adrenal gland of the frog.     

Host-graft relationships of isolated bovine chromaffin cells in rat periaqueductal grey

Summary The transplantation of chromaffin cells from the adrenal medulla into pain modulatory regions of the CNS has previously been shown to reduce pain sensitivity, most likely via local release of neuroactive substances from the transplanted cells. The ready availability of bovine adrenal glands, as well as the high levels of opioid peptides produced by their chromaffin cells, make these glands a potentially valuable donor source for antinociception studies. However, the success of these xenografts depends on their ability to survive and integrate within the host CNS.The aim of the present study was to assess host-graft relationships of bovine chromaffin cells transplanted to the rat CNS. We have found that isolated bovine chromaffin cells survive for at least three months in the rat periaqueductal grey, with no evidence of immunological response following a short-term course of immunosuppressant treatment. In the early stages following transplantation, only minor pathology is found at the injection site, which apparently recovers completely at later stages. The host-graft borders are not well demarcated, in contrast to solid tissue grafts.Neuronal processes of host origin, forming numerous synapses with the transplanted bovine chromaffin cells, are apparent by three weeks following transplantation. Migration also occurs from the graft into the host parenchyma, as evidenced by individual chromaffin cells found near host parenchymal blood vessels. The clusters of chromaffin cells found in the graft itself are generally not very vascular, in contrast to solid tissue grafts. The chromaffin cell clusters are surrounded by blood vessels of the non-fenestrated CNS type at the host-graft border. It is likely that the small size of the graft does not require extensive angiogenesis. The lack of fenestrated peripheral-type endothelial capillaries, normally seen in adrenal medullary tissue grafts, may contribute to the survival of these xenografts in the rat brain.     

Adrenal gland: structure, function, and mechanisms of toxicity

The adrenal gland is one of the most common endocrine organs affected by chemically induced lesions. In the adrenal cortex, lesions are more frequent in the zona fasciculata and reticularis than in the zona glomerulosa. The adrenal cortex produces steroid hormones with a 17-carbon nucleus following a series of hydroxylation reactions that occur in the mitochondria and endoplasmic reticulum. Toxic agents for the adrenal cortex include short-chain aliphatic compounds, lipidosis inducers, amphiphilic compounds, natural and synthetic steroids, and chemicals that affect hydroxylation. Morphologic evaluation of cortical lesions provides insight into the sites of inhibition of steroidogenesis. The adrenal cortex response to injury is varied. Degeneration (vacuolar and granular), necrosis, and hemorrhage are common findings of acute injury. In contrast, chronic reparative processes are typically atrophy, fibrosis, and nodular hyperplasia. Chemically induced proliferative lesions are uncommon in the adrenal cortex. The adrenal medulla contains chromaffin cells (that produce epinephrine, norepinephrine, chromogranin, and neuropeptides) and ganglion cells. Proliferative lesions of the medulla are common in the rat and include diffuse or nodular hyperplasia and benign and malignant pheochromocytoma. Mechanisms of chromaffin cell proliferation in rats include excess growth hormone or prolactin, stimulation of cholinergic nerves, and diet-induced hypercalcemia. There often are species specificity and age dependence in the development of chemically induced adrenal lesions that should be considered when interpreting toxicity data.     

Presence of microtubule-associated protein 2 in chromaffin cells

We have examined bovine adrenal chromaffin cells in culture for the presence of the microtubule-associated protein 2. Chromaffin cells could be identified in culture on the basis of their staining with antibodies directed against secretory granule proteins. Using immunoperoxidase staining microtubule-associated protein 2 immunoreactivity was demonstrated to be present in chromaffin cells but not fibroblasts in culture. Microtubule-associated protein 2 immunoreactivity was present in the cell body, processes and varicosities of the chromaffin cells. Microtubule-associated protein 2 polypeptides were shown to be present in an adrenal medullary homogenate but not chromaffin granule membranes by immunoblotting. The results indicate that the neuronal cytoskeletal polypeptide microtubule-associated protein 2 is present in adrenal chromaffin cells. The presence of microtubule-associated protein 2 in both neurons and chromaffin cells may be related to their common embryonic origin.     

Histogenesis of the human adrenal medulla. An evaluation of the ontogeny of chromaffin and nonchromaffin lineages.

The authors previously evaluated the expression of a panel of chromaffin-related genes during histogenesis of the human adrenal medulla. In these studies, chromaffin and nonchromaffin adrenal neuroblasts were identified. To better characterize these nonchromaffin neuroblasts, the authors evaluated two additional markers: HNK-1, an antibody recognizing the migratory neural crest cell; and S-100, a protein expressed by sustentacular cells of the adrenal medulla. HNK-1 immunoreactivity was found in both chromaffin and nonchromaffin cell types at different times during development, marking the nonchromaffin lineage during the second trimester of gestation as well as the chromaffin lineage in the neonatal period. In addition, S-100 expression was noted in some nonchromaffin neuroblasts, and sustentacular cells were first identified at approximately 28 weeks of gestational age. These data suggest a model of human adrenal medullary histogenesis that incorporates the chromaffin, ganglionic, and sustentacular lineages known to constitute the adult adrenal medulla.