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.     

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.     

Human fetal dopamine neurons grafted in a rat model of Parkinson's disease: ultrastructural evidence for synapse formation using tyrosine hydroxylase immunocytochemistry

Summary Human fetal mesencephalic dopamine (DA) neurons, obtained from 6.5–9 week old aborted fetuses, were grafted to the striatum of immunosuppressed rats with 6-hydroxydopamine lesions of the ascending mesostriatal DA pathway. The effects on amphetamine-induced motor asymmetry were studied at various timepoints after grafting. At eight weeks, functional graft effects were not evident but after 11 weeks small effects on motor asymmetry could be monitored and rats tested 19–21 weeks after grafting exhibited full reversal of the lesion-induced rotational behaviour. Four rats were sacrificed at different timepoints between 8 and 20 weeks and the grafted DA neurons were studied in tyrosine hydroxylase (TH) immunocytochemically stained sections at the light and electronmicroscopic level. The grafts contained a total of 500–700 TH-positive neurons in each rat. In one rat sacrificed 8 weeks after grafting the grafted neurons were TH-positive but exhibited virtually no fiber outgrowth. In another rat, sacrificed after 11 weeks, a sparse TH-positive fiber plexus was seen to extend into the adjacent host neostriatum. Two rats sacrificed after 20 weeks both contained TH-positive neurons that gave rise to a rich fiber network throughout the entire host neostriatum, and this fiber network was also seen to extend into the globus pallidus and nucleus accumbens. Very coarse TH-positive processes, identified as dendrites in the electron microscope, projected up to 1.5–2.0 mm from the graft into the host striatum. Ultrastructural analysis revealed that the grafted neurons had formed no TH-positive synaptic contacts with host striatal neurons after 8 weeks, and at 11 weeks some few TH-positive synapses were identified. Twenty weeks after transplantation, abundant TH-positive synaptic contacts with host neurons were seen throughout the neostriatum, and such contacts were identified in the globus pallidus as well. Thus, the present study provides tentative evidence for a time-link between the development of synaptic contacts and the appearance of functional graft effects. Similar to the normal mesostriatal DA pathway, ingrowing TH-positive axons formed symmetric synapses and were mainly seen to contact dendritic shafts and spines. However, in comparison to the normal rat striatum there was a higher incidence of TH-immunoreactive boutons forming synapses onto neuronal perikarya. The TH-positive dendrites that extended into the host striatum were seen to receive non-TH-immunoreactive synaptic contacts, presumably arising from the host neurons. These results suggest that human fetal DA neurons are able to develop a reciprocal synaptic connectivity with the host rat when grafted to the adult brain. Grafting of human fetal DA neurons may therefore be expected to provide a means of restoring regulated synaptic DA release in patients with Parkinson's disease.Some of the results of this study were presented at the Schmitt Neurological Sciences Symposium on Transplantation into the Mammalian CNS, Rochester, NY, USA, June 30, 1987     

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.     

Chronic implants of chromaffin tissue into the dopamine-denervated striatum. Effects of NGF on graft survival,fiber growth and rotational behavior

Summary Adult rat chromaffin tissue was transplanted into striatum of adult rat recipients whose nigrostriatal dopamine pathway had been lesioned on the grafted side by 6-hydroxydopamine. Long-term survival of the intrastriatal chromaffin grafts and the effects of treatment with nerve growth factor (NGF) was studied histochemically using Falck-Hillarp fluorescence histochemistry and functionally using rotational behavior induced by apomorphine. Small, cortex-free adrenal chromaffin tissue grafts survived permanently in striatum. The number of surviving cells was significantly increased by NGF. NGF treatment also caused transformation of many cells towards a more neuronal phenotype and greatly enhanced the adrenergic nerve fiber outgrowth into host brain tissue. NGF was either injected stereotaxically into the site of transplantation or infused continuously using implantable osmotic minipumps and a stereotaxically placed chronic indwelling dialysis fiber through striatum. The latter arrangement permitted continuous infusion of NGF for 14–28 days and caused a vigorous adrenergic nerve growth response by the grafts directed towards the source of NGF in the brain. There was a clearcut correlation between morphological signs of taking and rotational behavior. Grafts, and in particular grafts treated with NGF, were able to significantly and permanently counteract the rotational behavior induced by apomorphine. There seemed to be a dose relationship between NGF treatments and amount of reduction of asymmetric behavior. NGF treatment probably decreased the relative importance of diffuse release of catecholamines from chromaffin cells in the graft and increased the importance of adrenergic innervation of host striatum by cells in the graft. Immunofluorescence using antibodies against glial fibrillary acidic protein did not reveal any marked gliosis around the grafts nor were there any marked gliotic reactions around chronic indwelling dialysis fibers. We conclude that implantation of chromaffin tissue into striatum in conjunction with NGF treatments is an effective means of counteracting some of the symptoms of experimentally induced unilateral parkinsonism in rats.     

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.     

Efferent synaptic connections of dopaminergic neurons grafted into the caudate nucleus of experimentally induced parkinsonian monkeys are different from those of control animals

This study investigated the question of whether grafted dopamine cells in the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys form synapses and, if they do, whether their postsynaptic targets were the same as those in control monkeys or in previous studies in rats. Electron-microscopic single immunostaining was performed for tyrosine hydroxylase on vibratome sections prepared from the head of the caudate nucleus of controls and MPTP-treated African green monkeys (Cercopithecus aethiops sabaeus) that received a graft. Furthermore, correlated light- and electron-microscopic double immunostaining was carried out for tyrosine hydroxylase and calbindin in the same brain area of MPTP-treated plus grafted animals. In control monkeys, the majority (97%) of dopamine boutons terminate on spines that were also synaptic targets of immunonegative boutons forming asymmetric synaptic contacts: synaptic triads. In MPTP-treated, grafted animals, the majority of transplanted dopamine cells terminate on dendritic shafts (67%) and somata (32%), and only a few (1.33%) form axospine synapses. The results of the double immunostaining experiments indicated that these newly formed axosomatic and axodendritic synapses are associated with calbindin-immunoreactive, medium-sized, spiny striatonigral projection neurons. These observations indicate that: (1) dopamine from transplanted embryonic tissue acts via synaptic contacts on host neurons; (2) the primary synaptic targets of transplanted dopamine cells are not spines but dendrites and somata of host neurons; (3) these target neurons are the same as in control animals; and (4) comparing these observations with results of control and grafted rats, there are major species differences between rats and monkeys in the dopamine innervation of both control and transplanted animals. Received: 23 December 1997 / Accepted: 29 April 1998     

Long-term fate of neural precursor cells following transplantation into developing and adult CNS

Successful strategies for transplantation of neural precursor cells for replacement of lost or dysfunctional CNS cells require long-term survival of grafted cells and integration with the host system, potentially for the life of the recipient. It is also important to demonstrate that transplants do not result in adverse outcomes. Few studies have examined the long-term properties of transplanted neural precursor cells in the CNS, particularly in non-neurogenic regions of the adult. The aim of the present study was to extensively characterize the fate of defined populations of neural precursor cells following transplantation into the developing and adult CNS (brain and spinal cord) for up to 15 months, including integration of graft-derived neurons with the host. Specifically, we employed neuronal-restricted precursors and glial-restricted precursors, which represent neural precursor cells with lineage restrictions for neuronal and glial fate, respectively. Transplanted cells were prepared from embryonic day-13.5 fetal spinal cord of transgenic donor rats that express the marker gene human placental alkaline phosphatase to achieve stable and reliable graft tracking. We found that in both developing and adult CNS grafted cells showed long-term survival, morphological maturation, extensive distribution and differentiation into all mature CNS cell types (neurons, astrocytes and oligodendrocytes). Graft-derived neurons also formed synapses, as identified by electron microscopy, suggesting that transplanted neural precursor cells integrated with adult CNS. Furthermore, grafts did not result in any apparent deleterious outcomes. We did not detect tumor formation, cells did not localize to unwanted locations and no pronounced immune response was present at the graft sites. The long-term stability of neuronal-restricted precursors and glial-restricted precursors and the lack of adverse effects suggest that transplantation of lineage-restricted neural precursor cells can serve as an effective and safe replacement therapy for CNS injury and degeneration.     

Long-term fate of neural precursor cells following transplantation into developing and adult CNS

Successful strategies for transplantation of neural precursor cells for replacement of lost or dysfunctional CNS cells require long-term survival of grafted cells and integration with the host system, potentially for the life of the recipient. It is also important to demonstrate that transplants do not result in adverse outcomes. Few studies have examined the long-term properties of transplanted neural precursor cells in the CNS, particularly in non-neurogenic regions of the adult. The aim of the present study was to extensively characterize the fate of defined populations of neural precursor cells following transplantation into the developing and adult CNS (brain and spinal cord) for up to 15 months, including integration of graft-derived neurons with the host. Specifically, we employed neuronal-restricted precursors and glial-restricted precursors, which represent neural precursor cells with lineage restrictions for neuronal and glial fate, respectively. Transplanted cells were prepared from embryonic day-13.5 fetal spinal cord of transgenic donor rats that express the marker gene human placental alkaline phosphatase to achieve stable and reliable graft tracking. We found that in both developing and adult CNS grafted cells showed long-term survival, morphological maturation, extensive distribution and differentiation into all mature CNS cell types (neurons, astrocytes and oligodendrocytes). Graft-derived neurons also formed synapses, as identified by electron microscopy, suggesting that transplanted neural precursor cells integrated with adult CNS. Furthermore, grafts did not result in any apparent deleterious outcomes. We did not detect tumor formation, cells did not localize to unwanted locations and no pronounced immune response was present at the graft sites. The long-term stability of neuronal-restricted precursors and glial-restricted precursors and the lack of adverse effects suggest that transplantation of lineage-restricted neural precursor cells can serve as an effective and safe replacement therapy for CNS injury and degeneration.     

Chromaffin cell xenografts in the rat neocortex can produce antidepressive activity in the forced swimming test

Adrenal medullary allografts, as well as other monoaminergic tissues, have been demonstrated in our laboratory to increase antidepressive activity when transplanted into the frontal neocortex of rats. Refinement in the optimal parameters for xenograft viability has indicated that isolated bovine chromaffin cells may be an improved source of graft donor tissue. The aim of the present study was to determine whether isolated bovine chromaffin cell grafts to the rat frontal neocortex could provide an alternative source of catecholamines for antidepressant activity. Isolated bovine chromaffin cells, isolated bovine fibroblasts, or an equal volume of vehicle were unilaterally implanted into the right or left frontal cortex or right visual cortex. All rats were assessed before and 6 weeks after transplantation using the forced swimming test, a popular measure of antidepressant activity. Bovine chromaffin cell grafts in either the right or left frontal cortex produced significant increases in antidepressant activity compared to grafts of bovine fibroblasts and sham-operated or nontransplanted rats. In contrast, bovine chromaffin cells transplanted to the visual cortex did not affect antidepressant activity. Bovine fibroblast grafts in the frontal cortex also induced slight increases in antidepressant activity, although significantly less than chromaffin cell grafts. Morphological analysis revealed robust survival of tyrosine hydroxylase-positive chromaffin cells that retained their in situ ultrastructure and occasionally formed synaptic connections with the host parenchyma. These results suggest that xenografted isolated bovine chromaffin cells can provide a viable source of catecholamines for antidepressive activity.     

Up-regulation of ret by reserpine in the adult rat adrenal medulla

The receptor tyrosine kinase, ret, is activated by glial cell line-derived neurotrophic factor, neurturin and related ligands that bind to glycosylphosphatidylinositol-tailed receptors GFRalpha1-4. Ret expression is developmentally regulated and detectable only at very low levels in adult adrenal medulla. However, mutations of ret that cause constitutive activation or alter signal transduction give rise to adrenal medullary hyperplasia and pheochromocytomas in humans with hereditary multiple endocrine neoplasia (MEN) syndromes 2A and 2B and in animal models. These discordant observations pose the conundrum of how a molecule barely detectable in the adult adrenal can contribute to development of adrenal medullary pathology that typically occurs in adults. We recently reported that depolarization and phorbol esters that activate protein kinase C act synergistically with neurturin to up-regulate ret protein and mRNA expression in adult rat chromaffin cell cultures. Those findings suggested that ret expression in vivo is not static and might be regulated in part by neurally derived signals. We show here that the anti-hypertensive agent reserpine, which is known to cause a reflex increase in trans-synaptic stimulation of chromaffin cells, increases expression of ret mRNA and protein in adult rat adrenal medullary tissue in vivo. Elevated ret protein levels are detectable both by immunoblots and immunohistochemistry, which shows immunoreactive ret in chromaffin cells and neurons after reserpine administration. The finding that ret expression is subject to up-regulation by environmental signals in vivo suggests that epigenetic factors might influence the development of adrenal medullary disease by affecting the expression of ret. It is known that long-term administration of reserpine leads to the development of adrenal medullary hyperplasia and pheochromocytomas in rats. Our findings suggest potential utility of the rat model for studying the roles of ret in the adrenal medulla and the mechanisms of its involvement in MEN 2 and other pheochromocytoma syndromes.     

Long-term culture of human fetal spinal cord neurons: morphological, immunocytochemical and electrophysiological characteristics

Cultures were prepared from ventral spinal cord tissue from 8-11-week gestational human fetuses and grown for a period of up to 6 months. These cultures were studied by morphological, immunocytochemical and intracellular electrophysiological techniques. From 2 weeks in vitro and onward, small bipolar cells were found in outgrowths of spinal cord explants and were identified as neurons by positive immunoreactions with an antibody specific for neurofilament protein. In addition, a large population of glial fibrillary acidic protein-positive astrocytes and a smaller number of galactocerebroside-positive oligodendrocytes were recognized in these cultures. The development of synaptic terminals was also studied by electron microscopy. The first appearance of synaptic terminal was found in a 3-week culture and was an axo-dendritic synapse. During the next 2 months, there was a steady increase in number and structural maturation of synaptic profiles. In addition to axo-dendritic synapses, which were most common, axo-somatic and axo-axonic synapses were demonstrated. After 3 months in culture, the occurrence of large neurons possessing the characteristic features of mature neurons was also noted. Although the occurrence of oligodendrocytes in these cultures was confirmed, no myelination of axons was demonstrated by electron microscopy. Intracellular recordings were obtained from the cultured spinal cord cells, and these cells were identified clearly as neurons by their action potential responses to depolarizing current pulses. The average input resistance of these neurons was 31 M omega with resting membrane potential of -52 +/- 2.3 mV.     

Mixed cortical adenoma and composite pheochromocytoma-ganglioneuroma: an unusual corticomedullary tumor of the adrenal gland

Adrenal neoplasms composed of more than one cell type and demonstrating a mixed histologic appearance are exceedingly rare. We report the clinical and pathologic features of a morphologically distinctive tumor of the adrenal gland composed of cortical, chromaffin, and neural cells. Histologically, the tumor consisted of intermixed areas of proliferating cortical cells resembling adrenal cortical adenoma, neoplastic chromaffin cells consistent with pheochromocytoma, and a ganglioneuromatous stroma. The presence of the cortical, medullary, and neural components within the tumor was confirmed by immunohistochemical studies. The present case serves to broaden the morphologic spectrum of mixed tumors that may be encountered in the adrenal gland.     

Nerve growth factor receptor expression in peripheral and central neuroectodermal tumors, other pediatric brain tumors, and during development of the adrenal gland.

Nerve growth factor (NGF) is important to the survival, development, and differentiation of neurons. Its action is mediated by a specific cell surface transmembrane glycoprotein, nerve growth factor receptor (NGFR). In this study, NGFR expression by human fetal and adult adrenal medullary tissue, peripheral nervous system (PNS) neuroectodermal tumors (neuroblastoma, ganglioneuroblastoma, ganglioneuroma), pediatric primitive neuroectodermal tumors (PNETs) of the central nervous system (CNS), and CNS gliomas was examined by an immunohistochemical technique. Sixty-nine tumors in total were probed in this manner. Nerve growth factor receptor immunoreactivity was confined to nerve fibers and clusters of primitive-appearing cells in the fetal adrenal, and to nerve fibers and ganglion cells of the adult adrenal medulla; adrenal chromaffin cells were negative. In PNS neuroectodermal tumors, there was NGFR expression in tumor cells of 6 of 11 neuroblastomas and 6 of 6 ganglioneuroblastomas or ganglioneuromas. Thirteen of thirty-five CNS PNETs showed NGFR positivity. In most CNS PNETs, NGFR was restricted to scattered single or small groups of cells, but two tumors with astroglial differentiation showed much more extensive immunoreactivity. Most astrocytomas (11 of 14) and all ependymomas (3 of 3) were intensely NGFR positive.     

Differential occurrence and distribution of galanin in adrenal nerve fibres and medullary cells in rodent and avian species

The presence and distribution of galanin (GAL) in adrenal glands of rodent and avian species was investigated by light microscopic immunohistochemistry. GAL immunoreactivity was found in all medullary cells of guinea pig, duck and chicken adrenals. In contrast, only a subpopulation of medullary cells stained for GAL in Phodopus (Djungarian hamster) while the neuropeptide was completely missing in chromaffin cells of rat and pigeon. In rat, guinea pig and pigeon, GAL-immunoreactive nerve fibres were frequent in subcapsular regions and sparse in deeper cortical layers and in the chromaffin tissue. In contrast, only very few GAL fibres were found in Phodopus and no GAL fibres were observed in the adrenal glands of duck. In the chicken adrenal gland, fibres containing GAL were numerous throughout the organ and occurred in close vicinity to both steroidogenic as well as catecholaminogenic cells. The striking differences in the presence of GAL-positive cells and fibres are more pronounced between species within the rodent or avian group, respectively, than between the different vertebrate orders. The hitherto unknown and surprising variability of GAL expression and distribution in adrenal glands of various species suggests species-dependent functional (autocrine, paracrine and/or endocrine) roles of GAL in the neuroadrenal axis.     

Viability of adrenal chromaffin cells in the superior cervical ganglion of young adult and aged rats

Adrenal medullary tissue was autotransplanted to the superior cervical ganglion (SCG) of aged (26 months old) and young adult (3 months old) rats. Four and 20 weeks after operation, the viability of the transplants was evaluated using the formaldehyde-induced fluorescence (FIF) technique and tyrosine hydroxylase (TH) immunocytochemistry. Four weeks postgrafting, the transplant consisted of a densely-packed group of intensively fluorescent chromaffin cells in both age groups. The cells showed strong TH immunoreactivity and some of them were elongated, but only a few displayed short processes. At 20 weeks, most of the cells were spindle shaped and sent out fluorescent processes and a few of them were transformed toward ganglion-like cells. The results suggest that both young and old adrenal chromaffin cells are able to survive, produce neuronal processes and transform toward a neuronal phenotype in the rat superior cervical ganglion.     

Ultrastructural features of medullary chromaffin cell cultures

The ultrastructural organization on the fourth day of culture of chromaffin cells isolated from the bovine adrenal medulla was characterized based on electron microscopic and morphological analysis. We established that medullary chromaffin cells could be divided into four morphologically different subtypes. Most cells (49.1% of those examined) had a dense cytoplasm and fine dense granules. Cells with dense cytoplasm and large granules represented a second type of chromaffin cell (21.1%). Cells of the third type had a light cytoplasm, granules with a light halo and a well-developed Golgi apparatus (26.3%). The fourth type of chromaffin cell was characterized by moderately dense cytoplasm with well-expressed varicose rough endoplasmic reticulum (about 3.5%). Among concomitant cell types, cortical adrenal cells from the zona fasciculata and zona glomerulosa, epithelial cells, fibroblasts, lymphocytes, brown lipoblasts and glial Schwann cells were present. Morphological analysis implies that cells with dense cytoplasm and fine granules and those with light cytoplasm and haloed granules (75.4% in total) are adrenaline-containing cells, whereas the cells with dense cytoplasm and large granules (26.3%) contain noradrenaline. Cells with moderately dense cytoplasm and varicose reticulum share common morphological properties with classical glandular cells and, by their properties, were closer to noradrenaline-containing cells.It is concluded that chromaffin cells, which are the main cell type among cultured cells from adult bovine adrenal medulla, are morphologically quite heterogeneous. Other cell types of different nature may also be present in the culture and can locally influence the properties of the investigated medullary chromaffin cells used in electrophysiological experiments.     

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.     

Regional variations in the glial influence on synapse development in the mouse CNS

There is increasing evidence that synapse function depends on interactions with glial cells, namely astrocytes. Studies on specific neurons of the central nervous system (CNS) indicated that glial signals also control synapse development, but it remained unclear whether this is a general principle that applies to other neuronal cell types. To address this question, we developed new methods to immunoisolate neurons from different brain regions of postnatal mice and to culture them in a chemically defined medium. Electrophysiological recordings and immunocytochemical staining revealed vigorous synaptogenesis in hippocampal and cerebellar neurons, but not in retinal ganglion cells (RGCs) in the absence of glial cells. Co-culture with glia promoted synapse formation in RGCs as indicated by a strong increase in the incidence and frequency of action potential-independent miniature synaptic currents, but showed no such effects in hippocampal or cerebellar neurons. On the other hand, glial signals promoted the efficacy of excitatory synapses in all regions as indicated by an increase in the size of spontaneous synaptic events in cerebellar cultures and of miniature synaptic currents in hippocampal neurons and RGCs. Inhibitory synaptic currents remained largely unaffected by glia. Our results indicate that in the mammalian CNS, the way that glial signals promote the development of excitatory synapses depends on the type of neuron.     

Adrenal medulla calcium channel population is not conserved in bovine chromaffin cells in culture

During the stress response adrenal medullary chromaffin cells release catecholamines to the bloodstream. Voltage-activated calcium channels present in the cell membrane play a crucial role in this process. Although the electrophysiological and pharmacological properties of chromaffin cell calcium channels have been studied in detail, the molecular composition of these channels has not been defined yet. Another aspect that needs to be explored is the extent to which chromaffin cells in culture reflect the adrenal medulla calcium channel characteristics. In this sense, it has been described that catecholamine release in the intact adrenal gland recruits different calcium channels than those recruited during secretion from cultured chromaffin cells. Additionally, recent electrophysiological studies show that chromaffin cells in culture differ from those located in the intact adrenal medulla in the contribution of several calcium channel types to the whole cell current. However there is not yet any study that compares the population of calcium channels in chromaffin cells with that one present in the adrenal medulla. In order to gain some insight into the roles that calcium channels might play in the adrenal medullary cells we have analyzed the alpha1 subunit mRNA expression profile. We demonstrate that the expression pattern of voltage-dependent calcium channels in cultured bovine chromaffin cells markedly differs from that found in the native adrenal medulla and that glucocorticoids are only partially involved in those differences. Additionally, we show, for the first time, that the cardiac isoform of L-type calcium channel is present in both bovine adrenal medulla and cultured chromaffin cells and that its levels of expression do not vary during culture.