Adrenal medulla development and medullary-cortical interactions.

The mammalian adrenal gland is composed of two distinct tissue types in a bidirectional connection, the catecholamine-producing medulla derived from the neural crest and the mesoderm-derived cortex producing steroids. The medulla mainly consists of chromaffin cells derived from multipotent nerve-associated descendants of Schwann cell precursors. Already during adrenal organogenesis, close interactions between cortex and medulla are necessary for proper differentiation and morphogenesis of the gland. Moreover, communication between the cortex and the medulla ensures a regular function of the adult adrenal. In tumor development, interfaces between the two parts are also common. Here, we summarize the development of the mammalian adrenal medulla and the current understanding of the cortical-medullary interactions under development and in health and disease.

Cholinergic and peptidergic neurotransmission in the adrenal medulla: A dynamic control of stimulus-secretion coupling.

Synaptic neurotransmission at the splanchnic nerve-chromaffin cell synapse is a chief element of the stimulus-secretion coupling in the adrenal medullary tissue, managing and regulating the secretion of catecholamines. Making the state of play more intricate than initially envisioned, the synaptic vesicles of nerve terminals innervating the medulla contain various compounds, including various neurotransmitters and neuropeptides. Under basal conditions associated with a low splanchnic nerve discharge rate, neurotransmission is ensured by the synaptic release of the primary neurotransmitter acetylcholine (ACh). Under sustained and repetitive stimulations of the splanchnic nerve, as triggered in response to stressors, the synaptic release of neuropeptides, such as the pituitary adenylate cyclase-activating polypeptide PACAP, supplants ACh release. The anatomical and functional changes that occur presynaptically at the preganglionic splanchnic nerve, combined with changes occurring postsynaptically at nicotinic acetylcholine receptors (nAChRs), confer the adrenomedullary synapses a solid and persistent aptitude to functional remodeling, from birth to aging. The present review focuses on the composite cholinergic and noncholinergic nature of neurotransmission occurring at the splanchnic nerve-chromaffin cell synapse and its remodeling in response to physiological or pathological stimuli.

[EPIGENETIC REGULATION OF ADRENAL CHROMAFFIN CELLS DEVELOPMENT (REVIEW)].

Chromaffin cells of adrenal medulla have traditionally been considered as modified sympathetic neurons. However, the results of recent studies indicate the need to revise this concept. The article reviews recent findings in origin and ontogeny of adrenal chromaffin cells and transcriptional and posttranscriptional regulation of developmental processes. The article summarizes data on transcriptional control of chromaffin cells proliferation and maturation and participation of microRNA in regulation of chromaffin and sympathetic neuronal phenotype gene expression.

Localization and production of peptide endocannabinoids in the rodent CNS and adrenal medulla.

The endocannabinoid system (ECS) comprises the cannabinoid receptors CB1 and CB2 and their endogenous arachidonic acid-derived agonists 2-arachidonoyl glycerol and anandamide, which play important neuromodulatory roles. Recently, a novel class of negative allosteric CB1 receptor peptide ligands, hemopressin-like peptides derived from alpha hemoglobin, has been described, with yet unknown origin and function in the CNS. Using monoclonal antibodies we now identified the localization of RVD-hemopressin (pepcan-12) and N-terminally extended peptide endocannabinoids (pepcans) in the CNS and determined their neuronal origin. Immunohistochemical analyses in rodents revealed distinctive and specific staining in major groups of noradrenergic neurons, including the locus coeruleus (LC), A1, A5 and A7 neurons, which appear to be major sites of production/release in the CNS. No staining was detected in dopaminergic neurons. Peptidergic axons were seen throughout the brain (notably hippocampus and cerebral cortex) and spinal cord, indicative of anterograde axonal transport of pepcans. Intriguingly, the chromaffin cells in the adrenal medulla were also strongly stained for pepcans. We found specific co-expression of pepcans with galanin, both in the LC and adrenal gland. Using LC-MS/MS, pepcan-12 was only detected in non-perfused brain (∼ 40 pmol/g), suggesting that in the CNS it is secreted and present in extracellular compartments. In adrenal glands, significantly more pepcan-12 (400-700 pmol/g) was measured in both non-perfused and perfused tissues. Thus, chromaffin cells may be a major production site of pepcan-12 found in blood. These data uncover important areas of peptide endocannabinoid occurrence with exclusive noradrenergic immunohistochemical staining, opening new doors to investigate their potential physiological function in the ECS. This article is part of the Special Issue entitled 'Fluorescent Tools in Neuropharmacology'.

Fetal glucocorticoid synthesis is required for development of fetal adrenal medulla and hypothalamus feedback suppression.

During pregnancy, fetal glucocorticoid is derived from both maternal supply and fetal secretion. We have created mice with a disruption of the Cyp11a1 gene resulting in loss of fetal steroid secretion but preserving the maternal supply. Cyp11a1null embryos have appreciable although lower amounts of circulating corticosterone, the major mouse glucocorticoid, suggesting that transplacental corticosterone is a major source of corticosterone in fetal circulation. These embryos thus provide a means to examine the effect of fetal glucocorticoids. The adrenal in Cyp11a1 null embryos was disorganized with abnormal mitochondria and oil accumulation. The adrenal medullary cells did not express phenylethanolamine N-methyltransferase and synthesized no epinephrine. Cyp11a1 null embryos had decreased diencephalon Hsd11b1, increased diencephalon Crh, and increased pituitary Pomc expression, leading to higher adrenocorticotropin level in the plasma. These data indicate blunted feedback suppression despite reasonable amounts of circulating corticosterone. Thus, the corticosterone synthesized in situ by the fetus is required for negative feedback suppression of the hypothalamus-pituitary-adrenal axis and for catecholamine synthesis in adrenal medulla.

Developmental and stress-induced remodeling of cell­cell communication in the adrenal medullary tissue.

The adrenal medullary tissue contributes to maintain body homeostasis in reaction to stressful environmental changes via the release of catecholamines into the blood circulation in response to splanchnic nerve activation. Accordingly, chromaffin cell stimulus-secretion coupling undergoes temporally restricted periods of anatomo- functional remodeling in response to prevailing hormonal requirements of the organism. The postnatal development of the adrenal medulla and response to stress are remarkable physiological situations in which the stimulus- secretion coupling is critically affected. Catecholamine secretion from rat chromaffin cells is under a dual control involving an incoming initial command arising from the sympathetic nervous system that releases acetylcholine at the splanchnic nerve terminal-chromaffin cell synapses and a local gap junction-mediated intercellular communication. Interestingly, these two communication pathways are functionally interconnected within the gland and exhibit coordinated plasticity mechanisms. This article reviews the physiological and molecular evidence that the adrenal medullary tissue displays anatomical and functional adaptative remodeling of cell­cell communications upon physiological (postnatal development) and/or physiopathological (stress) situations associated with specific needs in circulating catecholamine levels.

Maternal perinatal undernutrition alters postnatal development of chromaffin cells in the male rat adrenal medulla.

Numerous data suggest that the development of the sympathoadrenal system is highly sensitive to the perinatal environment. We previously reported that maternal perinatal food restriction by 50% (FR50) altered chromaffin cell (CC) organization and activity in offspring at weaning. This study investigated the effects of FR50 on the postnatal time course of CC functional and structural adaptations. FR50 pups exhibited smaller and more abundant scattered clusters of noradrenergic CCs as early as postnatal day 7 (P7), indicating that morphological changes took place earlier during development. At birth, the adrenaline release was defective in FR50 pups, suggesting that maternal FR50 impaired the non-neurogenic control of catecholamine release. At P4, the catecholamine release in response to insulin-induced hypoglycaemia was also absent in FR50 pups. This was associated with the reduction of adrenal catecholamine contents, indicating that the failure to synthesize catecholamine might lead to impaired secretion. We hypothesized that maternal FR50 accelerated the functional connections between CCs and splanchnic nerve endings, leading to the premature loss of the non-neurogenic response. Acetylcholine-containing synaptic endings seemed more precociously functional in FR50 pups, as suggested by increased levels of acetylcholine esterase activity at P14. At P7, insulin-induced hypoglycaemia caused preferential adrenaline release associated with increased catecholamine contents in both groups. However, the response was accentuated in FR50 pups. At P14, the insulin challenge increased plasma levels of adrenaline in control rats, whereas it markedly enhanced the circulating level of both catecholamines in FR50 pups. We demonstrated that maternal FR50 leads to developmentally impaired noradrenergic CC aggregation and advanced splanchnic neurotransmission maturation associated with altered medulla activity in response to metabolic stress. This might contribute to the long-lasting malprogramming of the adrenal medulla and to the development of chronic adult diseases.

Acetylcholinesterase activity, choline acetyltransferase and vesicular acetylcholine transporter immunoreactivities in the rat adrenal gland during postnatal development.

From postnatal-day-0 to postnatal-day-2, a few acetylcholinesterase (AChE)-active and choline acetytransferase (ChAT)-immunoreactive nerve fibers and relatively numerous vesicular acetylcholine transporter (VAChT)-immunoreactive puncta were observed in the rat adrenal medulla. Despite relatively numerous clear vesicles in the nerve fibers, the synthesis and hydrolysis of acetylcholine may not be fully activated until postnatal-day-2. The number of AChE-active and ChAT-immunoreactive nerve fibers dramatically increased and that of VAChT-immunoreactive puncta gradually increased from postnatal-day-3 to postnatal-week-1. The synthesis and hydrolysis of acetylcholine may be dramatically activated in the nerve fibers of the medulla until postnatal-week-1. From postnatal-week-2 to postnatal-week-3, the number of AChE-active and the ChAT-immunoreactive nerve fibers gradually increased and reached the adult levels. The VAChT-immunoreactive puncta per unit area was maximum number at postnatal-week-2. The synthesis and hydrolysis of acetylcholine in the nerve fibers of the medulla may be completed between postnatal-week-2 to postnatal-week-3. The diameter of the VAChT-immunoreactive puncta gradually increased from postnatal-day-0 with aging. However, the number of the VAChT-immunoreactive puncta gradually decreased from postnatal-week-2 onwards. In electron-microscopy, the VAChT-immunoreactive deposits were seen in clusters of clear vesicles, and the diameter of the nerve fibers and the number of clear vesicles at postnatal-week-8 increased compared with those at postnatal-week-2. The AChE-active, ChAT-immunoreactive, and VAChT-immunoreactive nerve fibers observed around noradrenaline (NA) cells were denser than those around adrenaline (A) cells in the medulla at postnatal-week-8. These suggest that the preferential innervation of NA and A cells may cause the differential secretion NA and A.

Autocrine TGF-beta signaling is required for the GDNF/CNTF-induced neuronal differentiation of adrenal chromaffin tsAM5D cells expressing temperature-sensitive SV40 T-antigen.

We recently established adrenal medullary cell line tsAM5D, which was immortalized by use of a temperature-sensitive mutant of the oncogene simian virus 40 large T-antigen. In the present study, when co-treated with glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF), tsAM5D cells proliferated at the permissive temperature (33 degrees C) for the T-antigen expression and differentiated into neuron-like cells at the nonpermissive temperature (39 degrees C). Interestingly, in GDNF/CNTF-treated cultures, the addition of pan-specific transforming growth factor (TGF)-beta-neutralizing antibody did not affect the cell proliferation at 33 degrees C, but significantly reduced the survival of neuronally differentiated cells at 39 degrees C. Using real-time RT-PCR for analysis of GDNF/CNTF-treated cells, we found that the expression of mRNAs for TGF-beta1, TGF-beta2, and TGF-beta3 was up-regulated by the temperature shift. These results suggest that autocrine TGF-beta signaling is necessary for the survival of GDNF/CNTF-differentiated tsAM5D cells upon the temperature shift.

Identification and developmental changes of aromatase and estrogen receptor expression in prepubertal and pubertal human adrenal tissues.

CONTEXT: The mechanisms of postnatal adrenal zonation remain unclear. OBJECTIVE: To provide a clue for a possible role of estrogens in adrenarche, we studied the expression of estrogen receptor (ER)alpha, ERbeta, G protein-coupled receptor (GPR)30, and cP450aromatase (cP450arom) in human adrenal tissue. DESIGN: Human adrenal tissue was collected from three postnatal age groups (Grs): Gr 1, younger than 3 months (n = 12), fetal zone involution; Gr 2, 3 months to 6 yr (n = 17), pre-adrenarche; and Gr 3, older than 6-20 yr (n = 12), post-adrenarche period. RESULTS: ERbeta mRNA in Grs 1 and 3 was higher than in Gr 2 (P < 0.05). By immunohistochemistry and laser capture microdissection followed by RT-PCR, ERbeta was expressed in zona reticularis and fetal zone, GPR30 in zona glomerulosa (ZG) and adrenal medulla, while ERalpha mRNA and protein were undetectable. cP450arom mRNA in Gr 3 was higher than in Grs 1 and 2 (P < 0.05), and localized to ZG and adrenal medulla by laser capture microdissection. cP450arom Immunoreactivity was observed in adrenal medulla in the three Grs and in subcapsular ZG of Gr 3. Double-immunofluorescence studies revealed that cP450arom and chromogranin A only colocalize in adrenal medulla of subjects younger than 18 months. In these samples, exon 1.b-derived transcript was 3.5-fold higher, while exon 1.a-, 1.c-, and 1.d-derived transcripts were 3.3-, 1.9-, and 1.7-fold lower, respectively, than in subjects older than 6 yr. CONCLUSIONS: Our results suggest that estrogens produced locally in adrenal medulla would play a role in zona reticularis functional differentiation through ERbeta. The cP450arom and GPR30 expression in subcapsular ZG, colocalizing with a high-cell proliferation index, previously reported, suggests a local GPR30-dependent estrogen action in proliferation and migration of progenitor adrenal cells.

Endogenous ligands of PACAP/VIP receptors in the autocrine-paracrine regulation of the adrenal gland.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are the main endogenous ligands of a class of G protein-coupled receptors (Rs). Three subtypes of PACAP/VIP Rs have been identified and named PAC(1)-Rs, VPAC(1)-Rs, and VPAC(2)-Rs. The PAC(1)-R almost exclusively binds PACAP, while the other two subtypes bind with about equal efficiency VIP and PACAP. VIP, PACAP, and their receptors are widely distributed in the body tissues, including the adrenal gland. VIP and PACAP are synthesized in adrenomedullary chromaffin cells, and are released in the adrenal cortex and medulla by VIPergic and PACAPergic nerve fibers. PAC(1)-Rs are almost exclusively present in the adrenal medulla, while VPAC(1)-Rs and VPAC(2)-Rs are expressed in both the adrenal cortex and medulla. Evidence indicates that VIP and PACAP, acting via VPAC(1)-Rs and VPAC(2)-Rs coupled to adenylate cyclase (AC)- and phospholipase C (PLC)-dependent cascades, stimulate aldosterone secretion from zona glomerulosa (ZG) cells. There is also proof that they can also enhance aldosterone secretion indirectly, by eliciting the release from medullary chromaffin cells of catecholamines and adrenocorticotropic hormone (ACTH), which in turn may act on the cortical cells in a paracrine manner. The involvement of VIP and PACAP in the regulation of glucocorticoid secretion from inner adrenocortical cells is doubtful and surely of minor relevance. VIP and PACAP stimulate the synthesis and release of adrenomedullary catecholamines, and all three subtypes of PACAP/VIP Rs mediate this effect, PAC(1)-Rs being coupled to AC, VPAC(1)-Rs to both AC and PLC, and VPAC(2)-Rs only to PLC. A privotal role in the catecholamine secretagogue action of VIP and PACAP is played by Ca(2+). VIP and PACAP may also modulate the growth of the adrenal cortex and medulla. The concentrations attained by VIP and PACAP in the blood rule out the possibility that they act as true circulating hormones. Conversely, their adrenal content is consistent with a local autocrine-paracrine mechanism of action.

Regulation of alternative splicing of Slo K+ channels in adrenal and pituitary during the stress-hyporesponsive period of rat development.

Stress triggers release of ACTH from the pituitary, glucocorticoids from the adrenal cortex, and epinephrine from the adrenal medulla. Although functions differ, these hormone systems interact in many ways. Previous evidence indicates that pituitary and steroid hormones regulate alternative splicing of the Slo gene at the stress axis-regulated exon (STREX), with functional implications for the calcium-activated K+ channels prominent in adrenal medullary and pituitary cells. Here we examine the role of corticosterone in Slo splicing regulation in pituitary and adrenal tissues during the stress-hyporesponsive period of early rat postnatal life. The sharp drop in plasma corticosterone (CORT) that defines this period offers a unique opportunity to test CORT's role in Slo splicing. We report that in both adrenal and pituitary tissues, the percentage of Slo transcripts having STREX declines and recovers in parallel with CORT. Moreover, addition of 500 nm CORT to cultures of anterior pituitary cells from 13-, 21-, and 30-d postnatal animals increased the percentage of Slo transcripts with STREX, whereas 20 microm CORT reduced STREX representation. Applied to adrenal chromaffin cells, 20 microm CORT decreased STREX inclusion, whereas neither 500 nm nor 2 microm had any effect. The mineralocorticoid receptor antagonist RU28318 abolished the effect of 500 nm CORT on splicing in pituitary cells, whereas the glucocorticoid receptor antagonist RU38486 blocked the effect of 20 microm CORT on adrenal chromaffin cells. These results support the hypothesis that the abrupt, transient drop in CORT during the stress-hyporesponsive period drives the transient decline in STREX splice variant representation in pituitary, but not adrenal.

Maternal perinatal undernutrition alters neuronal and neuroendocrine differentiation in the rat adrenal medulla at weaning.

Epidemiological studies suggest that chronic adult diseases, such as type 2 diabetes and hypertension, can be programmed during fetal and early postnatal life. The nervous system regions governing vegetative functions and the hypothalamic-pituitary-adrenal axis are particularly sensitive to the perinatal nutritional status. Despite recent reports demonstrating that the activity of the sympathoadrenal system can be altered by early life events, the effects of maternal nutrient restriction on the adrenal medulla remain unknown. Using a rat model of maternal perinatal 50% food restriction (FR50) from the second week of gestation until weaning, immunohistochemical experiments revealed alterations in chromaffin cell aggregation and in nerve fiber fasciculation in the adrenal medulla of FR50 pups. These morphological changes were associated with enhanced circulating levels of catecholamines after decapitation (epinephrine by 55% and norepinephrine by 41%). Using macroarrays, we identified several genes whose expression was affected by maternal nutrient restriction. Semiquantitative RT-PCR confirmed the overexpression of four genes involved in neuroendocrine differentiation and neuronal plasticity (chromogranin B, growth-associated protein 43, neurofilament 3, and Slit2) in the adrenal glands of FR50 rats. Using in situ hybridization, we showed that these genes are solely expressed in the adrenal medulla. Together, our results suggest that perinatal maternal undernutrition markedly alters the differentiation of the adrenal medulla during postnatal life, resulting in enhanced activity of chromaffin cells at weaning. These alterations may persist in adulthood and participate to the programming of chronic adult diseases.

Butyrate, a gut-derived environmental signal, regulates tyrosine hydroxylase gene expression via a novel promoter element.

Butyrate is a diet-derived, gut fermentation product with an array of effects on cultured mammalian cells including inhibition of proliferation, induction of differentiation and regulation of gene expression. We showed that physiological concentrations of butyrate can regulate transcription of tyrosine hydroxylase (TH) and preproenkephalin (ppEnk) gene in PC12 cells. In promoter deletion studies, electrophoretic mobility shift assays and by site-directed mutagenesis, we identified a novel butyrate response element (BRE) in the 5' upstream region of the rat TH gene, homologous to the previously mapped motif in the ppEnk promoter. No such enhancers were found in DBH or PNMT promoters, and both catecholamine system-related gene promoters were unaffected by butyrate. The BRE motif interacts with nuclear proteins in a sequence-specific manner, shows binding potentiation in butyrate-differentiated PC12 cells and bound protein(s) are competed away with TH-CRE oligonucleotides or by the addition of CREB-specific antibodies, suggesting involvement of CREB or CREB-related transcription factors. Moreover, single point mutation in the distal BRE abolished binding of transcription factors and reduced the response to butyrate in transient transfection studies. The canonical CRE motif of the TH promoter was also found necessary for transcriptional activation of the TH gene by butyrate. Our data identified a novel functional element in the promoter of both the TH and ppEnk genes mediating transcriptional responses to butyrate. Dietary butyrate may have an extended role in the control of catecholamine and endogenous opioid production at the level of TH and ppEnk gene transcription neuronal plasticity, cardiovascular functions, stress adaptation and behavior.

Agrin mediates a rapid switch from electrical coupling to chemical neurotransmission during synaptogenesis.

In contrast to its well-established actions as an organizer of synaptic differentiation at the neuromuscular junction, the proteoglycan agrin is still in search of a function in the nervous system. Here, we report an entirely unanticipated role for agrin in the dual modulation of electrical and chemical intercellular communication that occurs during the critical period of synapse formation. When applied at the developing splanchnic nerve-chromaffin cell cholinergic synapse in rat adrenal acute slices, agrin rapidly modified cell-to-cell communication mechanisms. Specifically, it led to decreased gap junction-mediated electrical coupling that preceded an increase in nicotinic synaptic transmission. This developmental switch from predominantly electrical to chemical communication was fully operational within one hour and depended on the activation of Src family-related tyrosine kinases. Hence, agrin may play a pivotal role in synaptogenesis in promoting a rapid switch between electrical coupling and synaptic neurotransmission.

[Development of the human adrenal glands]. / Le développement de la surrénale humaine.

The human adrenal is an endocrine gland located at the superior part of the kidney. Composed of the adrenal cortex of mesoblastic origin and the adrenal medulla of neuroectoblastic origin, the human fetal adrenal grows considerably during the first three months of development. From 12 to 18 weeks of development (WD), the weight of the adrenals increases seven-fold. The gland's weight doubles from 18 to 28 WD and from 28 to 36 WD. At birth, the two adrenals weigh on average 10 g. At the 8th week, two zones are individualized in the adrenal cortex: the definitive zone and the fetal inner zone. At the second trimester, according to ultrastructural and biochemical studies, a third zone, called the transition zone, is individualized between the definitive zone and the fetal inner zone. The definitive zone persists, but the origin of the three zones (glomerular, fascicular and reticular) of adult adrenal cortex is not known. The fetal inner zone regresses from the 5th month of gestation and disappears totally one year after birth. At the 8th week, the immature neuroblasts migrate to the definitive zone, then to the fetal inner zone to compose the adrenal medulla, which develops essentially after birth and during the first year. Before the 10th week, the human fetal adrenal is able to produce steroid hormones, in particular dehydroepiandrosterone sulfate (DHEA-S); the secretion of cortisol remains discussed. The development of the human fetal adrenal is complex and is under the control of hormones (ACTH, LH and betaHCG), growth factors (ACTH essentially) and transcription factors (essentially SF1 and DAX-1). Knowledge of morphological and molecular phenomena of this development permits to understand the pathophisiology of congenital adrenal deficiencies.

Maternal adrenalectomy affects development of adrenal medulla.

This work investigates the effects of maternal adrenalectomy (ADX) on the development of the adrenal medulla. Adrenal catecholamines (AC) were measured at postnatal day (PN) 1, 8, 12 and 22 in rat offspring of ADX dams and in pups of control dams. The pups of ADX rats showed a reduction in AC concentrations in the adrenal medulla at PN 1, 12 and 22, although these were higher than in the pups of sham dams at PN 8. Further, in the pups of control mothers, there was an increase in ACs during the first two weeks of life whereas pups of ADX mothers only showed increases in noradrenaline, dopamine and adrenaline levels at day 8. These results suggest that maternal absence of corticosterone affects the medulla catecholamine content during development. These data support the idea that a maternal glucocorticoids are involved in the differentiation or/and maturation of the adrenal medulla.

Enhanced viability and neuronal differentiation of neural progenitors by chromaffin cell co-culture.

The transplantation of neural stem cells and progenitors has potential in restoring lost cellular populations following central nervous system (CNS) injury or disease, but survival and neuronal differentiation in the adult CNS may be insufficient in the absence of exogenous trophic support. Adrenal medullary chromaffin cells produce a trophic cocktail including basic fibroblast growth factor (FGF-2) and neurotrophins. The aim of this study was to evaluate whether chromaffin cells can provide a supportive microenvironment for neural progenitor cells. In order to assess this, the growth and differentiation of neural progenitor cell cultures from embryonic rat cortex were compared in standard FGF-2-supplemented neural progenitor growth media, in standard media but lacking FGF-2, or in media lacking FGF-2 but co-cultured with bovine chromaffin cells. Using bromodeoxyuridine (BrdU)-prelabeling, findings indicated poor survival of progenitor cultures in the absence of FGF-2. In contrast, the addition of chromaffin cells in co-culture appeared to 'rescue' the progenitor cultures and resulted in robust neurospheres containing numerous BrdU-labeled cells interspersed with and closely apposed to chromaffin cells. As indicated by H3 labeling, cells in co-cultures continued to proliferate, but at a substantially reduced rate compared with standard FGF-2 supplemented growth media. The co-cultures contained more beta-tubulin III-positive processes than parallel cultures maintained in FGF-2-supplemented media and these cells displayed a more mature phenotype with numerous varicosities and complex processes. These findings indicate that chromaffin cells can provide a supportive environment for the survival and neuronal differentiation of neural progenitor cells and suggest that their addition may be useful as a sustained source of trophic support to improve outcomes of neural stem cell transplantation.

G(z alpha) deficient mice: enzyme levels in the autonomic nervous system, neuronal survival and effect of genetic background.

Our laboratory has generated a genetically mutant mouse in which the alpha subunit of the heterotrimeric GTP binding protein, G(z) has been made dysfunctional by homologous recombination to determine its in vivo function. These animals show a characteristic failure to thrive phenotype. G(z alpha) is expressed in a variety of nervous system tissues as well as in the adrenal medulla. We therefore examined the autonomic nervous system of the G(z alpha) deficient mouse by measuring the activity of tyrosine hydroxylase and choline acetyltransferase in the superior cervical ganglia, submaxillary gland and the adrenal medulla. Preliminary results using animals of mixed BALB/c and C57BL/6 strains gave inconsistent results. Further experiments demonstrated differences in the activity of tyrosine hydroxylase and choline acetyltransferase between BALB/c and C57BL/6 mouse strains. The analysis of the pure strains showed a reduction in the size and enzyme levels of the adrenal gland and submaxillary glands of the G(z alpha) deficient mouse suggesting a role for adrenal insufficiency and/or nutritional disorders for the failure to thrive phenotype. The survival of sympathetic and sensory neurons was also examined in the G(z alpha) deficient mouse and in the presence of pertussis toxin, sympathetic but not sensory neuronal survival in G(z alpha) deficient mice was significantly attenuated. This suggests that in vivo other pertussis toxin sensitive G proteins may be recruited to compensate for the loss of G(z alpha).