Gene expression profiles of human melanoma cells with different invasive potential reveal TSPAN8 as a novel mediator of invasion.

BACKGROUND: Metastatic melanoma requires early detection, being treatment resistant. However, the earliest events of melanoma metastasis, and especially of dermal invasion, remain ill defined. RESULTS AND METHODS: Gene expression profiles of two clonal subpopulations, selected from the same human melanoma cell line, but differing in ability to cross the dermal-epidermal junction in skin reconstructs, were compared by oligonucleotide microarray. Of 26 496 cDNA probes, 461 were differentially expressed (>2-fold; P< 0.001), only 71 genes being upregulated in invasive cells. Among them, TSPAN8, a tetraspanin not yet described in melanoma, was upregulated at mRNA and protein levels in melanoma cells from the invasive clone, as assessed by RT-PCR, flow cytometry and western blot analysis. Interestingly, TSPAN8 was the only tetraspanin in which overexpression correlated with invasive phenotype. Flow cytometry of well-defined melanoma cell lines confirmed that TSPAN8 was exclusively expressed by invasive, but not non-invasive melanoma cells or normal melanocytes. Immunohistochemistry revealed that TSPAN8 was expressed by melanoma cells in primary melanomas and metastases, but not epidermal cells in healthy skin. The functional role of TSPAN8 was demonstrated by silencing endogenous TSPAN8 with siRNA, reducing invasive outgrowth from tumour spheroids within matrigel without affecting cell proliferation or survival. CONCLUSION: TSPAN8 expression may enable melanoma cells to cross the cutaneous basement membrane, leading to dermal invasion and progression to metastasis. TSPAN8 could be a promising target in early detection and treatment of melanoma.

[DNA microarray technology: principles and applications to the study of neurological disorders]. / Principes et intérêts pour l'étude des maladies neurologiques et technologie des puces ADN.

INTRODUCTION: DNA microarray is a powerful technology which can rapidly provide a high throughput and detailed view of the entire genome and transcriptome. In this review we discuss the basic principles behind gene expression microarrays, CGH arrays and DNA microarray genotyping, and their potential applications to neurological diseases. STATE OF THE ART: Microarray gene expression profiling is a reliable technology that has already been used with great success in the molecular classification of cancer. It is a very promising technology in the field of Neurooncology. One of the interesting characteristics of DNA microarrays is also that they can be used in a non-hypothesis-driven manner to discover new genomic characteristics that will enable to establish new pathophysiological hypotheses. Such a strategy has already yielded interesting new insights in the study of multiple sclerosis, Alzheimer disease or neuromuscular diseases. With DNA microarray genotyping it is now possible to detect mutations in many genes simultaneously. CONCLUSIONS: In Neurooncology DNA microarrays should help to establish a more accurate classification of brain tumors and recent studies have shown how gene expression profiling of brain tumors allows to uncover previously unrecognized patient subsets that differ in their survival. The applications of microarrays for the study of neurological diseases, like multiple sclerosis, Alzheimer disease or neuromuscular diseases are also promising both for generating new pathophysiological hypotheses and for enabling new molecular classifications. DNA microarray genotyping is a powerful technology that should help to discover genetic factors associated with multifactorial neurological disorders and help to diagnose complex neurogenetic diseases. This technology should also facilitate the realization of pharmacogenomic studies in neurological diseases.

Atrial fibrillation is associated with hypermethylation in human left atrium, and treatment with decitabine reduces atrial tachyarrhythmias in spontaneously hypertensive rats.

Atrial fibrillation (AF) is the most common cardiac arrhythmia. As the molecular mechanisms underlying the pathology are largely unknown, this cardiac arrhythmia remains difficult to treat. To identify specific molecular actors involved in AF, we have performed a transcriptomic analysis on left atrium (LA) from patients with valvular heart disease with or without AF. We showed that 1627 genes had altered basal expression level in LA tissue of AF patients compared with the control group. The significantly enriched gene ontology biological process "anatomical structure morphogenesis" contained the highest number of genes in line with changes in structure that occur when the human heart remodels following AF development (ie, LA dilatation and interstitial fibrosis). We then focused the study on Pitx2 (paired-like homeodomain 2), being the most altered transcription factor in LA from AF patients and from which compelling evidence have indicated that its reduced expression can be considered as a marker for the disease. In addition, its expression was inversely correlated with LA size. We demonstrated that AF is associated with Pitx2 promoter hypermethylation both in humans and arrhythmic aging spontaneously hypertensive rats. Chronic administration of a DNA methylation inhibitor (ie, 5-Aza-2'-deoxycitidine) improved ECG arrhythmic profiles and superoxide dismutase activities and reduced fibrosis in the left ventricle of spontaneously hypertensive rats. Taken together, these data support the notion that AF is associated with epigenetic changes in LA and provide a proof-of-concept that hypomethylating agents have to be considered in the treatment of atrial arrhythmias.

Gene expression profiling in insulinomas of Men1 beta-cell mutant mice reveals early genetic and epigenetic events involved in pancreatic beta-cell tumorigenesis.

Mutations of the MEN1 gene lead to the occurrence of multiple endocrine neoplasia type 1 (MEN1). To gain insights into the mechanisms of the tumorigenesis related to MEN1 inactivation, we have used mice in which the Men1 gene was specifically disrupted in pancreatic beta-cells. In these mice, we observed full penetrance of insulinoma with defined histological characteristics of tumorigenesis. To identify the genetic factors taking part in the tumour development, we performed gene expression profiling analysis of these insulinomas at different stages. Here, we show that in late stage insulinomas, 56 genes are up-regulated and 194 are down-regulated more than fourfold compared with normal pancreatic islets. Clustering analysis reveals the deregulation of Hox gene family and the genes involved in cell proliferation and cell cycle control. The altered expression of Igf2, Igfbp3 and Igfbp6 as well as cyclin A2, B2 and D2 are confirmed by quantitative RT-PCR, with the overexpression of all the three cyclins found in early stage insulinomas. Moreover, an increased proportion of cyclin A2- and D2-expressing cells and the overexpression of insulin-like growth factor 2 (IGF2) protein are detected in mouse Men1 insulinomas by immunostaining. Interestingly, the analysis of DNA methylation patterns by quantitative serial pyrosequencing reveals that four specific CpGs in the intragenic differentially methylated region 2 (DMR2) region of the Igf2 gene known to augment transcription through methylation are significantly hypermethylated in insulinomas of Men1 beta-cell mutant mice at 6 and 10 months of age, even before IGF2 overexpression can be detected. Thus, our data indicate the involvement of both genetic and epigenetic mechanisms in early tumorigenesis of beta-cells related to MEN1 inactivation.

Molecular cloning and sequencing of a cDNA encoding a beta-thyroid hormone receptor in muscovy duckling.

A cDNA clone encoding a beta-thyroid hormone receptor (TRbeta) from muscovy duckling liver was isolated and sequenced. Comparison with the chicken TRbeta sequence showed a high degree of homology. This cDNA was used as a probe to characterize the TRbeta mRNA transcripts expressed in muscovy duckling liver.

Differential regulation of uncoupling protein-1, -2 and -3 gene expression by sympathetic innervation in brown adipose tissue of thermoneutral or cold-exposed rats.

The control of uncoupling protein-1, -2 and -3 (UCP-1, UCP-2, UCP-3) mRNA levels by sympathetic innervation in rats was investigated by specific and sensitive RT-PCR assays. In rats reared at thermoneutrality (25 degrees C), unilateral surgical sympathetic denervation of interscapular brown adipose tissue (BAT) markedly reduced the UCP-1 mRNA level (-38%) as compared with the contralateral innervated BAT pad, but was without significant effect on UCP-2 and -3 mRNA levels. Cold exposure (7 days, 4 degrees C) markedly increased UCP-1 (+180%), UCP-2 (+115%) and UCP-3 (+195%) mRNA levels in interscapular BAT. Unilateral sympathetic denervation prevented the cold-induced rise in BAT UCP-1 and UCP-2 mRNAs, but not that in BAT UCP-3 mRNA. Results were confirmed by Northern blot analysis. These data indicate a differential endocrine control of UCP-1, UCP-2 and UCP-3 gene expression in rat BAT both at thermoneutrality and during prolonged cold exposure.

Catecholamine Metabolism in Locus Coeruleus Neurons: A Study of its Activation by Sciatic Nerve Stimulation in the Rat.

Sciatic nerve stimulation, which strongly activates noradrenergic locus coeruleus (NA-LC) neurons, was used in anaesthetized rats as a model to study the transneuronal control of catechol metabolism in this nucleus. We show, using in vivo electrochemistry and biochemical post-mortem assays, that a prolonged (20 min) unilateral sciatic nerve electrical stimulation led to a reversible enhancement (80 - 130%) of both endogenous and in vivo extracellular levels of 3,4-dihydroxyphenylacetic acid (DOPAC) within the contralateral LC region. An elevation in DOPAC levels was also observed in the ipsilateral nucleus but was always significantly lower. The response was abolished by a pretreatment with kynurenic acid, a non-selective excitatory amino acid (EAA) antagonist known to block footshock-induced excitations of NA-LC neurons: in antagonist-treated rats, the stimulation induced a non-significant effect (+ 30%) on endogenous DOPAC levels, which contrasted with the highly significant effect (+ 113%) observed in vehicle-treated animals. As the major source of EAA afferents to the LC originates in the nucleus paragigantocellularis, we made an attempt to suppress activation by a section of these fibres. An incision performed obliquely (45 degrees ) between LC and PGi greatly and significantly attenuated, but did not totally suppress, the increase in DOPAC endogenous content due to the stimulation. These experiments indicate that a peripheral stimulus provokes an activation of catecholamine metabolism within the soma - dendritic region of the NA-LC cells. They suggest that this effect may be mediated, at least in part, by afferent pathways originating from the medulla which utilize an EAA as transmitter.

Tissue-specific modulation of rat glucagon receptor mRNA by thyroid status.

The influence of thyroid status on glucagon receptor mRNA levels was investigated in rats using a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Glucagon receptor mRNA was detected in liver, brown and white adipose tissues (BAT and WAT) and brain. In BAT and WAT, pharmacologically-induced moderate hypothyroidism resulted in a marked reduction in the relative abundance of glucagon receptor mRNA. Short-term treatment of hypothyroid rats with exogenous 3,3',5'-triiodo-L-thyronine (T3), resulting in a marked hyperthyroidism, reversed the phenomenon in BAT while the reversal was only partial in WAT. In the liver, there was no significant effect of mild hypothyroidism while there was a positive effect of hyperthyroidism. In brain, the relative tissue abundance of glucagon receptor mRNA was not affected by the large changes in plasma T3. The present results therefore indicate that thyroid status may modulate the relative abundance of glucagon receptor mRNA in a tissue-specific manner.

Lack of glucocorticoids enhances the early activation of the medullary catecholaminergic cell groups triggered by restraint stress.

We have investigated whether the stress-induced activation of the medullary catecholaminergic neurons, that was shown previously to provide the main central activation input to the hypothalamo-pituitary-adrenocortical axis during an immobilization stress, is sensitive to circulating corticosteroids. Experiments were carried out on adrenalectomized rats that were first maintained on corticosterone in the drinking water for 5 days following surgery and then switched to corticosterone-free water 15 h before stress application. Some of the latter animals were injected with dexamethasone. Activation of the brainstem Catecholaminergic neurons was estimated by assaying 3,4-dihydroxyphenylacetic acid (DOPAC), a side metabolite of the noradrenaline and adrenaline biosynthesis pathway that was established previously as a reliable index of the activity of these neurons. In the so-called A(1) C(1) and A(2) C(2) Catecholaminergic groups of the medulla, lack of corticosterone under these experimental conditions did not modify the basal level of DOPAC but led to a further enhancement (+ 35% to 40%) of the approximately 2-fold increase in DOPAC content observed 15 min after the onset of the 5-min immobilization stress. No significant further enhancement of the stress-induced DOPAC increase was observed in the locus coeruleus. In these adrenalectomized rats, dexamethasone pretreatment prevented the enhancement of the stress-induced increase in DOPAC level observed in the medullary cell groups but did not abolish the response to stress. Lack of endogenous corticosteroids led to a 10-fold enhancement of the adrenocorticotropin increase following immobilization stress. Pretreatment with dexamethasone fully abolished the stress-induced increase in adrenocorticotropin plasma level. Our results show that circulating corticosteroids reduce the stress-induced activation of the medullary A(1) and A(2) C(2) groups i.e. those that contribute mainly to the catecholaminergic innervation of the hypothalamo-pituitary-adrenocortical axis within the hypothalamic paraventricular nuclei. However, since the feedback regulation of the central catecholaminergic systems is much less efficient than the feedback actually observed on the adrenocorticotropin secretion, we suggest that it is likely to play a minor physiological role in the overall feedback regulation exerted by circulating corticosteroids on the hypothalamo-pituitary-adrenocortical axis.

Hemorrhage-Induced Activations of Adrenocorticotropin Release and Catecholamine Metabolism in the Ventrolateral Medulla are Differently Affected by Glucocorticoid Feedback.

We have compared the effects of increasing doses of dexamethasone on the hemorrhage-induced stimulation of the corticotropic axis and the metabolism of the catecholamines of the A1 group in the ventrolateral medulla. Adrenocorticotropin was measured in sequential samples of plasma while the metabolism of the catecholamines was recorded by in vivo electrochemistry in urethane-anesthetized rats. Combined intracerebroventricular injection of specific adrenergic blockers (α(1) -antagonist, prazosin and ß-antagonist, propranolol) prevented the stimulation of the adrenocorticotropin release by hemorrhage. Pretreatment with dexamethasone (1 mg/kg sc) fully blocked the hemorrhage-induced adrenocorticotropin release but did not affect the concomitant stimulation of the catecholamine metabolism in A1 cells. The latter was partially decreased only with the highest dose (10 mg/kg sc). While a central catecholaminergic input appears to be necessary for the hemorrhage-induced stimulation of the corticotropic axis, it does not seem to play a significant role in the feedback regulation by glucocorticoids.

The habituation of brainstem catecholaminergic groups to chronic daily restraint stress is stress specific like that of the hypothalamo-pituitary-adrenal axis.

It has previously been shown that immobilization and ether stress induce activation of the hypothalamo-pituitary-adrenal (HPA) axis and that this activation occurs subsequent to activation of brain stem catecholaminergic neurones. In the present study we have investigated whether the brain stem catecholaminergic (CA) neurons show habituation to chronic daily intermittent exposure to the same restraint stress comparable to that of the HPA axis. The level of activity of the brainstem CA groups was estimated by measurement in tissue punches of content of 3,4-dihydroxyphenylacetic acid (DOPAC), a side metabolite of noradrenaline and adrenaline biosynthesis which has been shown to be a reliable index of the stress-induced activation of the CA groups. The level of activity of the HPA axis was determined by measurement of plasma corticosterone and adrenocorticotropic hormone (ACTH) levels. The animals were submitted to a 15 min restraint stress daily. They were sacrificed at the end of the stress session on day 3, 5 and 10. The ACTH response to the acute restraint stress whilst unchanged on day 3 was significantly decreased on day 5 (-54%) and day 10 (-70%) compared to the response in naive rats. The approximately twofold increase in DOPAC level induced by acute restraint stress in the so-called CA medullary group A1/C1 of naive rats was reduced in daily restraint rats on day 5 (-22%) and day 10 (-30%) but was unchanged on day 3. A small (-20%) decrease of the stress-induced DOPAC response in the A2/C2 CA group and locus coeruleus was also observed on day 10.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of catecholaminergic neurons in the ventral medulla by various stressors monitored with in vivo electrochemistry.

Catecholaminergic metabolism in the A1 cell group of the ventrolateral medulla oblongata was followed after application of 3 interoceptive stressful stimuli by monitoring the extracellular concentration of 3,4-dihydroxyphenylacetic acid (DOPAC) with an in vivo voltammetric approach. These stimuli provoked an increase of the DOPAC signal with different time course and amplitude. Histamine led to a maximal 200% increase that vanished within two hours. Insulin induced a long-lasting increase of up to 350% that could be reversed by glucose infusion. Electrical stimulation of the sciatic nerve triggered an immediate increase of up to 140% which stopped with the ending of the stimulation. The time-course of this activation is compatible with a possible involvement of catecholaminergic afferents from the A1 group projecting to the paraventricular nucleus in the stimulation of the hypothalamic neurosecretory cells elicited by the stressors.

Effects of gestational hypoxia on mRNA levels of Glut3 and Glut4 transporters, hypoxia inducible factor-1 and thyroid hormone receptors in developing rat brain.

Alterations of brain development result from noxious intrauterine signals, as oxygen deprivation, which decrease glucose energetic yield. To verify the hypothesis that a defect of brain energetic adaptation is responsible for these alterations, we have studied the effects of gestational hypoxia (10% oxygen during the last 2 weeks of fetal life) on cerebral ontogenesis of glucose transporters which control the limiting step of glucose utilization by neurons. This study is realised in rats by quantification of whole brain Glut3 and Glut4 mRNA in 14- and 19-day-old embryos (E14, E19), newborn (P0) and 7 postnatal-day-old rats (P7) by using reverse transcription-polymerase chain reaction (RT-PCR) method. We have associated our study with the analysis of a transcriptional factor, the hypoxia inducible factor-1alpha (HIF-1alpha), known to control the expression of glucose transporter, and with a family of transcriptional factors, the thyroid hormone receptors (TR), regulating specific genes involved in brain development. The data show (1) for the first time the Glut4 and HIF-1alpha gene expression in fetal rat brain which are detected as soon as E14, (2) that gestational hypoxia induces an increase of mRNA transcript levels of Glut3, Glut4, TRalpha2, TRbeta1 and HIF-1alpha genes mainly or exclusively at E14, and (3) that the absence of response of Glut3 and HIF-1alpha at E19 in hypoxic vs. normoxic group could indicate an insufficient energetic adaptation at this period of development which could lead to the neural alterations observed postnatally.

Central noradrenergic reactivity to stress in Maudsley rat strains.

Maudsley reactive (MR) and Maudsley nonreactive (MNRA) rats were submitted to a single session of acute 5-min immobilization stress and immediately sacrificed by decapitation. Subsequent neurochemical analysis revealed an elevation of 3,4-dihydroxyphenylacetic acid levels in the locus coeruleus and in the ventrolateral medulla, but not in the dorsomedial medulla, of rats of the two strains compared with nonstressed controls. This response was greater in the MR than in the MNRA group, suggesting a strain difference in the reactivity of the central noradrenergic cells to acute stress.

Characterization of a cDNA encoding an alpha thyroid hormone receptor in muscovy duckling.

A complementary deoxyribonucleic acid (cDNA) clone encoding an alpha thyroid hormone receptor (TR alpha) from muscovy duckling liver was isolated and sequenced. Comparison with the chicken TR alpha sequence showed a high degree of homology. Despite 45 nucleotide substitutions, the deduced peptide sequence was similar. This cDNA was used as a probe to characterize the TR alpha mRNA transcripts expressed in muscovy duckling liver and skeletal muscle.

Genome-wide expression analyses establish dendritic cells as a new osteoclast precursor able to generate bone-resorbing cells more efficiently than monocytes.

Dendritic cells (DCs), mononuclear cells that initiate immune responses, and osteoclasts (OCs), multinucleated bone-resorbing cells, are hematopoietic cells derived from monocytic precursor cells. Using in vitro generated dendritic cells, we previously showed that human and murine DCs could transdifferentiate into resorbing osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL). In this study we globally compared by transcriptomic profiling this new osteoclast differentiation pathway from DCs with the canonical differentiation pathway from monocytes. DNA chip data revealed that starting from two very distinct cell types, treatment with M-CSF and RANKL generated two highly similar types of osteoclast. In particular, DC-derived osteoclasts expressed all the characteristic marker genes of monocyte-derived osteoclasts. Two major molecular events could be observed during osteoclastogenesis: downregulation of a large set of monocyte or DC specific markers, together with upregulation of characteristic osteoclast marker genes. Most interestingly, our transcriptomic data showed a closer molecular profile between DCs and OCs than between monocytes and OCs. Our data establish DCs as a new osteoclast precursor able to generate OCs more efficiently than monocytes.

Developmental plasticity of the carotid chemoafferent pathway in rats that are hypoxic during the prenatal period.

The chemoreflex pathway undergoes postnatal maturation, and the perinatal environment plays a critical role in shaping respiratory control system. We investigated the role of prenatal hypoxia on the maturation of the chemoreflex neural circuits regulating ventilation in rat. Effects of hypoxia (10% O2) from the 5th to the 20th day of gestation were studied on male offspring at birth and on postnatal days 3, 7, 21 and 68. Maturation of the respiratory control system was assessed by in vivo tyrosine hydroxylase (TH) activity measurement in peripheral chemoreceptors (carotid bodies, petrosal ganglia), and in brainstem catecholaminergic cell groups (A2C2c and A1C1 areas in the medulla, A5 and A6 areas in the pons). Resting ventilation and ventilatory response to hypoxia were evaluated as functional sequelae. In peripheral structures, prenatal hypoxia reduced TH activity within the first postnatal week and enhanced it later. In contrast, in central areas, prenatal hypoxia upregulated TH activity within the first postnatal week and downregulated it later. The in vivo TH activity impairment is therefore tissue specific, with an opposite effect on the peripheral and central neural circuits. A shift of the effect of prenatal hypoxia occurred between 1 and 3 weeks, indicating a postnatal temporal effect of prenatal hypoxia. An important period in the development of the chemoafferent pathway occurred between the first and the third postnatal week. Functionally, prenatal hypoxia impaired resting ventilation and ventilatory response to hypoxia. The alterations of the catecholaminergic components of the chemoafferent pathway resulting from prenatal hypoxia might contribute to impair postnatal respiratory behaviour.

Differential time course activation of the brain stem catecholaminergic groups following chronic adrenalectomy.

The activity of the brain stem catecholaminergic (CA) cell groups of the ventrolateral (A1C1) and dorsomedial (A2C2) medulla that are known to contain primarily nonadrenergic neurones (A1 and A2) and a smaller proportion of adrenergic cells (C1 and C2) as well as the noradrenergic group locus ceruleus (LC) in the dorsal pons was determined at various times up to 16 days following surgical adrenalectomy. The activity of the CA cell groups was estimated by the rate of tyrosine hydroxylation in vivo that was assessed by measuring the 3.4-dihydroxyphenylalanine (DOPA) accumulated 20 min following administration of DOPA decarboxylase inhibitor NSD 1015. In the medullary nuclei noradrenaline content was found around 40- up to 70-fold the adrenaline content. This result was taken as evidence that the noradrenergic cells are likely to provide the main contribution to the tyrosine hydroxylation rate that we measured. Endogenous DOPA content represented between 2 and 10% of the noradrenaline content. NSD 1015 induced an accumulation of DOPA that was linear for at least 20 min and reached at this time more than 10-fold the endogenous level. While no modification of the in vivo tyrosine hydroxylation rate was observed in the LC, a significant increase was found in both medullary groups following adrenalectomy. In the A1C1 group it was detected 8 days after surgery and was then maintained with a maximum that represented up to a 60% increase over the basal value. In the A2C2 group the activation was slightly delayed and less marked. Increase in ACTH level occurred much earlier: it was about 70% of the maximal level already 4 days following adrenalectomy.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential early time course activation of the brainstem catecholaminergic groups in response to various stresses.

The effects of various stressors (restraint, ether, histamine and insulin-induced hypoglycemia stress) on the early time course activation of the different catecholaminergic (CA) cell groups A1/C1, A2/C2 and locus ceruleus (LC) from the brainstem were studied. The activity of the central noradrenergic neurons was assessed by measuring in tissue punches the 3,4-dihydroxyphenylacetic acid (DOPAC) level, a side metabolite of noradrenaline (NA) and adrenaline biosynthesis that is thought to reflect the activity of NA cells. Short 5 min restraint stress led to an immediate increase of DOPAC level in the three CA groups. In the A1/C1 and A2/C2 groups the maximal increase, respectively +75 and +50%, was already reached at the end of the application of the stress while for the LC the maximum (+84%) was obtained 15 min after the onset of the stress. Return to baseline level was achieved within 2 h. Continuous immobilization stress did not further alter the DOPAC concentration in the LC and the A1/C1 while a progressive increase up to 85% in the A2/C2 group was seen over 20 min. Following a 2-min exposure to ether, DOPAC was increased in all three structures within 5 min. At this time the maximum was already reached in the A1/C1 and LC, respectively +99 and +43%. After histamine or insulin injection DOPAC level increased in the A1/C1 and A2/C2 in the +25/+50% range but was not significantly affected in the LC. In all the stress situations studied the increase in DOPAC level, particularly in the A1/C1 group always preceded or was concomitant to the increase of plasma corticosterone.(ABSTRACT TRUNCATED AT 250 WORDS)

The involvement of noradrenergic ascending pathways in the stress-induced activation of ACTH and corticosterone secretions is dependent on the nature of stressors.

The aim of the present study was to explore in male rats the role of the catecholaminergic innervation of the hypothalamus in corticotropic and adrenal responses to different kinds of stress conditions. For this purpose, 6-hydroxydopamine (3 micrograms in 0.2 microliter saline) was stereotaxically and bilaterally infused at two levels of the main noradrenergic ascending brain stem bundle (NAB-X). The efficiency of catecholaminergic denervation of the hypothalamus was checked by measuring noradrenaline concentrations in paraventricular nuclei punches by HPLC and was confirmed by a 86% fall in noradrenaline levels of NAB-X rats killed after the stress experiments. Seven days after lesioning the NAB, sham operated controls and NAB-X lesioned animals were divided into 4 groups and submitted to 4 different stressors, i.e.: 2 min ether vapors (n = 5), 1 h immobilization (n = 7), i.v. histamine (2 mg/kg; n = 7) or i.v. insuline (10 I.U./kg; n = 8) injections. ACTH and corticosterone were measured in blood samples sequentially taken from a chronic carotid cannula, before stress and at short intervals over the 2 following hours. In comparison to the respective control groups, NAB-X dramatically reduced the ACTH response to ether (-78%) and to restraint (-53%) stress whereas the corticosterone response was affected to a lesser extent. In contrast, NAB-X slightly altered these responses in the histamine-treated group, although, surprisingly, the ACTH response tended to decrease and that of corticosterone to increase. Finally, NAB-X provoked a biphasic response to insuline-induced hypoglycemia, with a very early (5 min) rise in ACTH and corticosterone in comparison to the control group, followed by a trend to low hormonal levels up to 120 min. These results strongly suggest a differential involvement of the hypothalamic noradrenergic innervation upon the hypothalamic-pituitary-adrenal axis according to the nature of stress conditions.