THE EFFECTS OF METOPIRONE AND ADRENALECTOMY ON THE REGULATION OF THE ENZYMES MONOAMINE OXIDASE AND CATECHOL-O-METHYL TRANSFERASE IN DIFFERENT BRAIN REGIONS

Abstract— The role of glucocorticoids in the regulation of the enzymes monoamine oxidase (MAO) and catechol- O -methyltransferase (COMT) in brain regions has been studied. Glucocorticoids were blocked by Metopirone. The activities of MAO and COMT were determined in the hypophysis, hypothalamus, pineal gland and in the rest of brain. All the cerebral tissues except the pineal gland demonstrated highest MAO activity 8 h after Metopirone administration, when glucocorticoids were at the lowest level. Prolonged treatment for 10 days significantly augmented MAO activity in brain, hypophysis and hypothalamus, and COMT in the hypophysis increased by 56 per cent. The COMT activity in the rest of the brain did not change significantly with either short or prolonged administration. Complete ablation of the adrenal cortex resulted in a 167 per cent rise in MAO activity of the hypophysis. Metopirone and hydrocortisone inhibit MAO and COMT in vitro. The results suggest that glucocorticoids in the circulation of normal animals inhibit the activities of MAO and COMT. The inhibition or ablation of these hormones removes this rate-limiting control of catecholamine degradation resulting in higher activities of MAO and COMT. Metopirone, an inhibitor of MAO and COMT in vitro , acts in the opposite direction in vivo due to its inhibitory effects on corticoid biosynthesis.     

5,7-Dihydroxytryptamine: Regional Brain Concentrations Following Intraventricular Administration to Rats

The concentrations of 5,7-dihydroxytryptamine (5,7-DHT) and serotonin (5-HT) were measured in brainstem, hypothalamus and cerebral cortex 0, 2, 6, 12, and 24 hours following the bilateral, lateral ventricular injection of 5,7-DHT (100 g/each ventricle) into adult male rats. At 6 hours, 5,7-DHT levels had decreased 99% from 0 hr values in all brain regions. Thereafter, 5,7-DHT levels continued to decline in cortex, but not in hypothalamus or brainstem; at 24 hr, but not 48 hr, 5,7-DHT peaks were still measurable in each brain region examined. Serotonin levels in all three regions also fell markedly by 2-6 hours after 5,7-DHT administration. At 24 hours, hypothalamus and brainstem 5HT levels had declined >70% and cerebral cortex 50% below control values. The relevance of these findings to the protective action of monoamine reuptake blockers is discussed.     

THE EFFECT OF AGE, GROWTH RETARDATION AND ASPHYXIA ON ASCORBIC ACID CONCENTRATIONS IN DEVELOPING BRAIN

Abstract— The concentration of ascorbic acid in whole rat brain during the first week of postnatal life was up to 100 per cent higher than in adult animals. A progressive fall in concentration occurred between 4 and 30 days of age. Corresponding changes did not occur in liver and adrenal gland, two other organs rich in ascorbic acid. Rats subjected to growth retardation during the fetal and suckling periods had, at 25 days of age, levels of ascorbic acid in the cerebellum and brainstem significantly higher than those of control animals. A period of prolonged asphyxia in 5-day-old rats resulted in a significant 4 per cent reduction in whole brain ascorbic acid concentration.     

ACETYL- AND BUTYRYLCHOLINESTERASE ACTIVITY OF SELECTED BRAIN AREAS IN DEVELOPING RATS AFTER NEONATAL X-IRRADIATION*

Acetylcholinesterase and butyrylcholinesterase activities in sensori-motor cortex, hypothalamus, cerebellum, and brain stem were compared in normally developing Long-Evans rats and after neonatal whole-body exposure to 450 r X-radiation. Enzyme activities were measured on three postnatal days: day 10, when brain is still immature; day 24, when it has reached functional and morphological maturity; and day 64, after sexual maturation. In controls, acetylcholinesterase and butyrycholinesterase activities increased with age in all areas, especially between 10 and 24 days; e.g., in sensori-motor cortex acetylcholinesterase activity increased 60 per cent from 10 to 24 days and 12 per cent from 24 to 64 days. At all ages acetylcholinesterase activity was highest in the brain stern, followed in decreasing order by the hypothalamus, cerebellum, and sensori-motor cortex. Butyrylcholinesterase activity was higher in subcortical than in cortical areas. In neonatally irradiated rats, acetylcholinesterase activity was significantly decreased in the ontogenetically newer structures at 10, but not at 64, days; in the hypothalamus, it remained normal at 10 days but was significantly decreased at 24 and 64 days. Butyrylcholinesterase activity was significantly decreased in some areas 1 week after radiation but returned to normal at 24 days. Total esterase activity in whole blood was signtficantly decreased at 10 days in irradiated rats but returned to control levels by the end of the experiment. The greatest post-radiation decline in acetylcholinesterase activity (60 per cent below controls) did not result in spontaneous gross behaviour alterations, but may be related to disturbances in functional brain maturation evidenced by specific tests. If the role of acetycholine as a central neurotransmitter is accepted, these data suggest that radiation alters acetycholine/acetylcholinesterase ratios and thereby cholinergie synaptic transmission.     

Effects of cortisol treatment on brain and adrenal corticotropin-releasing hormone (CRH) content and other parameters regulated by CRH

Corticotropin-releasing hormone (CRH) has been found in both hypothalamic and extrahypothalamic sites of the brain and also in the adrenal medulla. To study the timing and location of delayed glucocorticoid action in rats, we measured the effects of 2-day and 7-day cortisol treatment on immunoreactive CRH concentrations in hypothalamus, cerebral cortex, hippocampus, cerebellum, and adrenal gland. The activity of the hypothalamo-pituitary-adrenal (HPA) axis and the sympathoadrenal system were also measured. Studies were carried out both in the afternoon and/or in the morning, to get information about possible circadian changes. CRH contents were not changed in any brain areas studied, except there was a trend of decrease in the hypothalamus compared to vehicle in the afternoon due to the lack of circadian increase after 7-day cortisol treatment. Pituitary ACTH content decreased significantly after 7-day treatment, while beta-endorphin did not. Plasma levels of ACTH, corticosterone, norepinephrine and epinephrine and adrenal ACTH and beta-endorphin contents decreased after 2-day, adrenal CRH content after 7-day treatment with cortisol. Our findings suggest, that chronic cortisol treatment inhibits the circadian activation of the HPA axis at all levels but has variable effects on baseline measures because it causes different changes in release and synthesis at different sites.     

Regional physiological adaptation of the central nervous system deiodinases to iodine deficiency

The goal of the present investigation was to analyze the types 2 (D2) and 3 (D3) iodothyronine deiodinases in various structures within the central nervous system (CNS) in response to iodine deficiency. After 5-6 wk of low-iodine diet (LID) or LID + 2 microg potassium iodide/ml (LID + KI; control), rats' brains were processed for in situ hybridization histochemistry for D2 and D3 mRNA or dissected, frozen in liquid nitrogen, and processed for D2 and D3 activities. LID did not affect weight gain or serum triiodothyronine, but plasma thyroxine (T4) was undetectable. In the LID + KI animals, D3 activities were highest in the cerebral cortex (CO) and hippocampus (HI), followed by the olfactory bulb and was lowest in cerebellum (CE). Iodine deficiency decreased D3 mRNA expression in all CNS regions, and these changes were accompanied by three- to eightfold decreases in D3 activity. In control animals, D2 activity in the medial basal hypothalamus (MBH) was similar to that in pituitary gland. Of the CNS D2-expressing regions analyzed, the two most responsive to iodine deficiency were the CO and HI, in which an approximately 20-fold increase in D2 activity occurred. Other regions, i.e., CE, lateral hypothalamus, MBH, and pituitary gland, showed smaller increases. The distribution of and changes in D2 mRNA were similar to those of D2 activity. Our results indicate that decreases in the expression of D3 and increases in D2 are an integral peripheral component of the physiological response of the CNS to iodine deficiency.     

A study of selected catecholamine metabolizing enzymes: a comparison of depressive suicides and alcoholic suicides with controls

—The regional distributions of monamine oxidase (MAO) (EC 1.4.3.4), catechol-O-methyltransferase (COMT) (EC 2.1.1.6), tyrosine hydroxylase (TH) (EC 1.14.3.2), and dopamine-β-hydroxylase (DBH) (EC 1.14.2.1) have been examined in human brains obtained at autopsy from persons who died of natural causes (controls), and from persons who committed suicide and were further categorized as suffering from affective disorder (depression) or from alcoholism. Post mortem animal studies showed no changes in MAO or COMT activities in rabbit brain or in DBH activity in rat brain when the intact bodies were left at room temperature up to 24 h. TH activity in rabbit brains, however, began to decline immediately after death and after 24 h at room temperature it was approximately 48 per cent of the fresh brain level. There was no significant variation in activity of COMT, TH and DBH in human brain attributable to age or sex. MAO activities in the 60–70 yr decade were 34 per cent higher than in the 30–40 yr decade. MAO activities were highest in the hypothalamus and substantia nigra, TH activities were highest in substantia nigra, putamen and head of caudate, and DBH activities were greatest in tegmentum of pons and hypothalamus. Only minimal regional differences in COMT activities were observed. No significant differences were found between enzyme activities in brain areas of controls and suicides with the possible exception of TH in the substantia nigra, where the depressive suicides (but not the alcoholics) showed greater activity (P < 0·02). These findings appear not to support the catecholamine hypothesis of affective disorder.     

Effect of stress on the acetylcholinesterase activity of the hypothalamus-pituitary-adrenal axis in the rat

Acetylcholinesterase (AchE, EC 3.1.1.7) activity was determined in cerebral cortex, hypothalamus, adenohypophysis and adrenal gland in response to acute and chronic stress. Chronic exposure of animals to cold stress (at 4 degrees C for 7 days) resulted in significant decline of AchE activity in all tissues studied. Similar results were obtained when animals were exposed to acute immobilization and cold stress (at 4 degrees C) simultaneously. In another experiment, animals were treated with 2 mg/kg of corticosterone prior to AchE determination. Corticosterone administration resulted in a significant decline in AchE activity of the cortex, the hypothalamus and the adrenal but failed to affect the adenohypophysis AchE level. Exposing adrenalectomized animals to acute stress resulted in no significant changes in the cortex and the hypothalamus but caused a significant decline in AchE of the adenohypophysis. It was concluded from this study that corticosterone might mediate the stress effect on AchE activity.     

Effects of Electroconvulsive Shock on Tetrahydrobiopterin and GTP-Cyclohydrolase Activity in the Brain and Adrenal Gland of the Rat

The effects of a single and repeated electroconvulsive shock (ECS) (300 mA, 0.2 s) on tetrahydrobiopterin (BH4) levels and GTP-cyclohydrolase activity in the brain and adrenal glands of rats were examined. Twenty-four hours after the last ECS treatment (one/day for 7 days), biopterin levels were significantly elevated in the locus coeruleus, hippocampus, frontal cortex, hypothalamus, ventral tegmental area, and adrenal gland. There were no changes in biopterin levels after a single application of ECS. GTP-cyclohydrolase activity was significantly increased in the locus coeruleus, frontal cortex, hippocampus, hypothalamus, and adrenal gland 24 h after repeated ECS and remained elevated in certain tissues up to 8 days after the last treatment. Kinetic analysis of adrenal and locus coeruleus GTP-cyclohydrolase 1 day after 7 days of ECS showed significant changes in both Km and Vmax values. These data suggest that the long-term increases in BH4 levels and GTP-cyclohydrolase activity after repeated ECS may play a part in the mediation of the antidepressant effects of ECS.     

Stress-Related Changes in Cerebral Catecholamine and Indoleamine Metabolism: Lack of Effect of Adrenalectomy and Corticosterone

The concentrations of catecholamine and indoleamine metabolites were measured in intact and adrenalectomized mice to determine whether adrenal hormones mediate or modulate the stress-induced responses. Thirty minutes of footshock resulted in significant increases of the ratios of the dopamine (DA) catabolite, dihydroxyphenylacetic acid (DOPAC), to DA in prefrontal cortex, nucleus accumbens, striatum, hypothalamus, and brainstem, and of homovanillic (HVA)/DA ratios in nucleus accumbens, striatum, amygdala, and hypothalamus. Ratios of 3-methoxy-4-hydroxyphenylethyleneglycol to norepinephrine (NE) were also increased in prefrontal cortex, nucleus accumbens, septum, amygdala, hypothalamus, hippocampus, and brainstem. The concentration of NE was decreased in amygdala. 5-Hydroxyindoleacetic acid (5-HIAA)/5-hydroxytryptamine (5-HT, serotonin) ratios and free tryptophan were also increased in every brain region. Very similar data were obtained from mice restrained for 30 min. Adrenalectomy resulted in increased HVA/DA ratios in prefrontal cortex and striatum, and 5-HIAA/5-HT in septum. The stress-related changes were largely similar in adrenalectomized mice. Significant interactions between adrenalectomy and footshock treatment occurred in prefrontal cortical DOPAC/DA and hypothalamic NE which was depleted only in adrenalectomized mice, suggesting tendencies for these measures to be more responsive in adrenalectomized mice. Corticosterone administration (0.5-2.0 mg/kg s.c.) which resulted in plasma concentrations in the physiological range did not alter the concentrations of the cerebral metabolites measured in any region. We conclude that adrenal hormones do not mediate cerebral catecholamine or indoleamine metabolism in stress, although adrenalectomy may affect HVA and 5-HIAA metabolism, and there was a tendency for catecholamines to be more sensitive to stress in adrenalectomized animals.     

Effect of Acute Hypoxia on Ascorbate Content of Plasma, Cerebral Cortex, and Adrenal Gland

Levels of ascorbic acid (AA) in the plasma, brain, and adrenal gland of rats were determined after 15 min of hypoxia (PaO2 less than 25 mm Hg) and following asphyxia. In rabbits, AA plasma levels were followed up to 75 min of reoxygenation following 15 min of hypoxia of the same severity. A significant increase (approximately 70%) in AA levels was found in plasma of rats and rabbits after hypoxia and asphyxia. This increase was found to be transient, with a return to normal levels within 1 h after resumption of normal oxygenation. Pretreatment with dexamethasone reduced the increase in AA level in both rabbits and rats. Adrenalectomy in rats, performed 24 h before the experiment, abolished the response to hypoxia. Ascorbate levels in the cerebral cortex, hypothalamus, and adrenal gland of awake rats subjected to hypoxia or asphyxia were found to be the same as in normoxic rats. Our results suggest that the observed changes in plasma AA levels are probably mediated through adrenocorticotropic hormone and that the adrenal gland is the major source of ascorbate efflux into the circulation during oxygen deprivation.     

Ganglion cells immunoreactive for catecholamine-synthesizing enzymes,neuropeptide Y and vasoactive intestinal polypeptide in the rat adrenal gland

Immunohistochemistry has been used to demonstrate tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) immunoreactivities, and acetylcholinesterase (AChE) activity was demonstrated in rat adrenal glands. The TH, DBH, NPY and VIP immunoreactivities and AChE activity were observed in both the large ganglion cells and the small chromaffin cells whereas PNMT immunoreactivity was found only in chromaffin cells, and not in ganglion cells. Most intraadrenal ganglion cells showed NPY immunoreactivity and a few were VIP immunoreactive. Numerous NPY-immunoreactive ganglion cells were also immunoreactive for TH and DBH; these cells were localized as single cells or groups of several cells in the adrenal cortex and medulla. Use of serial sections, or double and triple staining techniques, showed that all TH- and DBH-immunoreactive ganglion cells also showed NPY immunoreactivity, whereas some NPY-immunoreactive ganglion cells were TH and DBH immunonegative. NPY-immunoreactive ganglion cells showed no VIP immunoreactivity. AChE activity was seen in VIP-immunopositive and VIP-immunonegative ganglion cells. These results suggest that ganglion cells containing noradrenaline and NPY, or NPY only, or VIP and acetylcholine occur in the rat adrenal gland; they may project within the adrenal gland or to other target organs. TH, DBH, NPY, and VIP were colocalized in numerous immunoreactive nerve fibres, which were distributed in the superficial adrenal cortex, while TH-, DBH- and NPY-immunoreactive ganglion cells and nerve fibres were different from VIP-immunoreactive ganglion cells and nerve fibres in the medulla. This suggests that the immunoreactive nerve fibres in the superficial cortex may be mainly extrinsic in origin and may be different from those in the medulla.     

Differential Roles of Raphe Nuclei in the Regulation of Dopamine β-Hydroxylase in the Adrenal Gland of the Rat

The effect of reserpine on the activity of dopamine beta-hydroxylase (DBH) in the adrenal gland of the rat was determined following electrolytic lesion of the dorsal raphe nucleus (DRN) or medial raphe nucleus (MRN). In sham-operated rats, as well as in those with a lesion of the DRN, there was no significant modification of the action of reserpine on this enzyme. However, a lesion of MRN potentiated the inducing action of the drug. A specific role of MRN in the serotonergic regulation of adrenal DBH is suggested by this work.     

Age-dependent changes of nucleic acid labeling in different rat brain regions

The effects of aging on in vivo DNA and RNA labeling and on RNA content in various brain regions of 4-, 12-, and 24-month-old rats were investigated. No difference in [methyl-¹⁴C]thymidine incorporation into DNA of cerebral cortex and cerebelllum during aging was observed.The ratio of RNA/DNA content significantly decreased from 4 to 24 months of age in cerebral cortex, cerebellum and striatum. RNA labeling decreased by 15% in cerebral cortex of 24-month-old animals while in the other brain areas examined (cerebellum, hippocampus, hypothalamus, brainstem, striatum) did not change during aging.In the cerebral cortex, the ratio of the specific radioactivity of microsomal RNA to that of nuclear RNA, determined by in vivo experiments, was not affected by the aging process. A significant decrease of total, poly(A)+ RNA and poly(A)- RNA content was observed in the same brain area of 24-month-old rats compared to 4-month-old ones. Moreover, densitometric and radioactivity patterns obtained by gel electrophoresis of labeled RNA after in vitro experiments (tissue slices of cerebral cortex) showed a different ribosomal RNA processing during aging. In vivo chronic treatment with CDP-choline was able to increase RNA labeling in corpus striatum of 24-month-old animals.     

PROTEOLYTIC ENZYMES AND EXPERIMENTAL DEMYELINATION IN THE RAT AND MONKEY

Abstract— Visible lesions from monkeys with acute experimental allergic encephalomyelitis (EAE) induced by injection of purified myelin basic protein were assayed for acid proteinase, for a neutral proteinase at pH 6·5, and one lesion was measured for cathepsin A. Acid proteinase was increased to 152–176 per cent of levels in normal-appearing brain areas, neutral proteinase increased to 220–258 per cent, and the one lesion assayed for cathepsin A was 840 per cent of control. These enzymes were measured in the brain stem of Lewis rats with acute EAE as a result of basic protein injection and compared to Freund's adjuvant-injected controls. Acid proteinase was increased significantly to an average level of 128 per cent of control, the increase in neutral proteinase was not significant, and cathepsin A levels were 258 per cent of control, a highly significant increase. The rise in cathepsin A levels was not seen until the onset of paralytic symptoms. The brain stem of Wistar rats treated with whole spinal cord which show EAE in a milder form than the Lewis rat did not contain significantly higher enzyme levels than the control. The increases in acid proteinase and cathepsin A in brain stems were compared to levels of these enzymes in lymph nodes of EAE, Freund's adjuvant-injected controls and uninjected controls. The level of acid proteinase of lymph nodes/g protein did not change appreciably in the course of EAE development in the Lewis and Wistar rats and was about 3–4 times the activity in the brain stem. The cathepsin A in the inguinal lymph nodes of Wistar and Lewis rats injected with whole spinal cord in Freund's adjuvant increases to a level 2× that of the lymph nodes of the uninjected control. The cathepsin A levels in these activated lymph nodes was 6–8 × that of the control brain stem. The lymph nodes of Lewis and Wistar rats injected with Freund's adjuvant alone showed the same increase in cathepsin A as those from rats injected with spinal cord. The brain stem of rats undergoing severe demyelination as a result of chronic administration of triethyl tin did not show the enzyme increases. These results are compatible with the theory that proteolytic enzyme increases in EAE (and probably multiple sclerosis) are due to the invasion of mononuclear cells, some of which are probably lymphocytes. Whether or not these enzymes participate in the actual dissolution of myelin is unknown.     

Effects of orexin on cultured porcine adrenal medullary and cortex cells

New orexigenic peptides called orexins have recently been described in the neurons of the lateral hypothalamus and perifornical area. No orexins have been found in the adipose tissues or visceral organs, including the adrenal gland. However, expression of the orexin receptor (OXR) in the rat adrenal gland has been reported. With regard to the effects of orexins on peripheral organs, we previously reported that orexins suppress catecholamine synthesis and secretion in the rat pheochromocytoma cell line PC12. To further clarify the pharmacological effects of orexins on peripheral organs, we examined the effects of orexin-A on catecholamine, cortisol, and aldosterone secretion, using cultured porcine adrenal glands. We initially confirmed the expression of the orexin receptor (OXR-1) in cultured porcine adrenal medulla and cortex. Orexin-A (1000 nM) significantly increased the release of both epinephrine (E) and norepinephrine (NE) from porcine adrenal medullary cells. Similarly, orexin-A (> or = 100 nM) significantly increased the release of both cortisol and aldosterone from porcine adrenal cortex cells. Orexin-A (100 nM) significantly inhibited basal and the PACAP-induced increase in cAMP levels in adrenal medullary cells. Conversely, orexin-A (>o = 100 nM) significantly increased the cAMP level in adrenal cortex cells. These results indicate that orexin-A induces the release of catecholamine from porcine adrenal medullary cells, and aldosterone and cortisol from the cortex cells and has opposite effects on cAMP levels in adrenal medulla and cortex.     

EVIDENCE FOR EXTRANORADRENERGIC DOPAMINE-β-HYDROXYLASE ACTIVITY IN RAT SALIVARY GLAND

—Three days after superior cervical ganglionectomy of adult Sprague-Dawley rats, the levels of endogenous norepinephrine, the uptake process for [³H]norepinephrine and the activity of tyrosine hydroxylase decreased 99 per cent in the ipsilateral salivary gland. In contrast, the activity of dopamine-β-hydroxylase and DOPA decarboxylase fell to 30 per cent of the activity of the contralateral innervated gland. Examination of the cofactor requirements, the characteristics of activation by cupric ion and the immunologic identity of this residual hydroxylase activity indicated that it was authentic dopamine-β-hydroxylase. The residual dopamine-β-hydroxylase in the denervated gland had the same subcellular distribution as the enzyme in the innervated salivary gland. Procedures that caused atrophy or hypertrophy of the acinar cells did not affect the total content of dopamine-β-hydroxylase in the denervated salivary gland. Chemical sympathectomy with 6-hydroxy-dopamine caused a 40 per cent decrement in the serum levels of dopamine-β-hydroxylase but a 30 per cent increase in its activity in the denervated salivary gland. Although denervation caused a complete loss of endogenous norepinephrine in the salivary gland, it resulted in only a 15 per cent decrement in the levels of endogenous octopamine and β-phenylethanolamine, two other products of dopamine-β-hydroxylase.     

Inhibition of Histamine Synthesis in Brain by α-Fluoromethylhistidine, a New Irreversible Inhibitor: In Vitro and In Vivo Studies

a-Fluoromethylhistidine (α-FMH), a new potent inhibitor of histidine decarboxylase (HD), has been used for in vitro and in vivo studies of brain HD. Following a preincubation with (+)-α-FMH, brain HD activity was inhibited in a time-dependent and concentration-dependent manner. The enzyme activity was not restored by overnight dialysis against standard buffer. The (–) antimer of α-FMH was ineffective. When injected intraperitoneally in a single dose of 20 mg/kg, (±)-α-FMH induced a complete loss in HD activity in cerebral cortex and hypothalamus as well as in peripheral tissues, such as stomach. At a dosage of 100 mg/kg (±)-α-FMH did not alter histamine-N-methyltransferase, DOPA decarboxylase, and glutamate decarboxylase activities. The maximal decrease of HD activity occurred after 2 h in both cerebral cortex and hypothalamus, but the time course of the recovery of enzyme activity was slower in the cerebral cortex. The enzyme activity reached control value within 3 days in hypothalamus and was not fully restored after 4 days in cerebral cortex. Contrasting with the diminished HD activity, a substantial concentration of histamine remained present in five regions of mouse brain. Thus, α-FMH is a highly specific irreversible inhibitor of brain HD activity and its efficacy makes it useful to study the physiological role of brain histamine.     

Ghrelin-containing neuron in cerebral cortex and hypothalamus linked with the DVC of brainstem in rat

Ghrelin is a newly discovered brain-gut peptide and an endogenous ligand for growth hormone secretagogues receptor (GHS-R). Ghrelin and GHS-R present extensively in central and peripheral tissues such as stomach, brain and other organs of rodent and human, which suggest it has multiple biological effects. It has been reported that ghrelin has significant role in the regulation of energy homeostasis, food intake and appetite. The organization of central circuitry appears to play an important role in integrating orexigenic effects of ghrelin, but the detail is not fully clear. In this study, we examined the expression of ghrelin, ghrelin mRNA and GHS-R mRNA in cerebrum and brainstem by RT-PCR and immunofluorescence histochemistry, and analyzed the connection among the cerebral cortex, hypothalamus, dorsal vagal complex (DVC). The results showed that the positive staining of ghrelin was found on the pyramidal neuron of layer V in the sensorimotor area of cerebral cortex, cingulate gyrus, as well as in the neuron of lateral hypothalamus (LH), PVN and ARC. The expression of ghrelin mRNA and GHS-R mRNA were also found in the sensorimotor cortex and hypothalamus by method of RT-PCR. The GHS-R mRNA was also found in the DVC of medulla oblongata. Other finding is that the FG/ghrelin dual labeled neurons were found in LH of hypothalamus (not in cortex). The ghrelin-containing neuron in the LH projects its axon to the DVC with the method of retrograde tracing. In conclusion, the ghrelin neurons are located not only in hypothalamus (LH, PVN, ARC), but also in the cortex (sensorimotor area, cingular gyrus), and the fibers of ghrelin neurons in hypothalamus projected directly to the DVC. It suggests that ghrelin plays its role from hypothalamus to brainstem as a neurotransmitter or neuromodulator to regulate function of vagal nuclei in brainstem.     

Neuropeptide W is expressed in the noradrenalin-containing cells in the rat adrenal medulla

Neuropeptide W (NPW) is an endogenous ligand for GPR7, a member of the G-protein-coupled receptor family. NPW plays an important role in the regulation of both feeding and energy metabolism, and is also implicated in modulating responses to an acute inflammatory pain through activation of the hypothalamus-pituitary-adrenal axis. GPR7 mRNA has been shown to be expressed in the hypothalamus, pituitary gland and adrenal cortex. Similarly, NPW expression has been demonstrated in the brain and pituitary gland. However, the precise distribution of NPW-producing cells in the adrenal gland remains unknown. The aim of this study was to explore the distribution and localization of NPW immunoreactivity in the rat adrenal gland. Total RNA was prepared from the hypothalamus, pituitary gland and adrenal gland. RT-PCR revealed the expression of NPW mRNA in these tissues, while in situ hybridization demonstrated the presence of NPW mRNA in the adrenal medulla. When immunohistochemistry was performed on sections of adrenal gland, NPW-like immunoreactivity (NPW-LI) was observed in the medulla but not in the cortex. Moreover, NPW-LI was found to be co-localized in cells which expressed dopamine beta hydroxylase but not phenylethanolamine-N-methyltransferase. The finding that NPW is expressed in noradrenalin-containing cells in the adrenal medulla suggests that it may play an important role in endocrine function in the adrenal gland.