Photosynthetic characteristics of summer maize under different planting patterns and the responses to nitrogen application of previous crop / Características fotossintéticas do milho verão sob diferentes padrões de plantio e as respostas à aplicação de nitrogênio da cultura anterior

Maize (Zea mays L.) is one of the most important grain crops in the North China Plain. Management practices affect the photosynthetic characteristics and the production of summer maize. This two-year (2014-2015) study examined the effects of different planting patterns and the application of nitrogen to previous winter wheat (Triticum aestivum L.) on the photosynthetic characteristics, yield and radiation use efficiency (RUE) of summer maize. Field experiments used a two-factor split-plot design with three replicates at Taian, Shandong Province, China (36°09' N, 117°09' E). The experiments involved two planting patterns(ridge planting, RP; and uniform row planting, UR) and two nitrogen application levels of previous winter wheat (N1, 112.50 kg ha-1; N2, 225.00 kg ha-1). The results indicated that the application of nitrogen on previous crop and ridge planting of the following crop had significant effects on the photosynthetic characteristics and yields of summer maize. Compared with UR, this study found that RP increased the chlorophyll content index (CCI), leaf area index (LAI), net photosynthetic rate (Pn), dry matter (DM), yield and grain RUE by 4.1%, 6.3%, 5.2%, 6.4%, 8.9% and 9.4%, respectively. The CCI, LAI, Pn, yield, and grain RUE of N2 were 9.7%, 3.3%, 3.7%, 10.0% and 10.1% higher than those of N1, respectively. RP combined with the application of nitrogen on previous crop of winter wheat could increase the CCI, LAI, Pn, DM, ultimately increasing the grain yield and RUE of the following summer's maize. It was concluded that previous crop nitrogen application and RP pattern treatment resulted in optimal cropping conditions for the North China plain.

O milho (Zea mays L.) é uma das culturas de grãos mais importantes da Planície do Norte da China. Práticas de manejo afetam as características fotossintéticas e a produção do milho verão. Este estudo de dois anos (2014-2015) examinou os efeitos de diferentes padrões de plantio e a aplicação de nitrogênio ao trigo de inverno anterior (Triticum aestivum L.) sobre as características fotossintéticas, produtividade e eficiência de uso de radiação (RUE) do milho verão. Experimentos de campo usaram um delineamento em parcelas subdivididas de dois fatores com três repetições em Taian, província de Shandong, China (36°09' N, 117°09' E). Os experimentos envolveram dois padrões de plantio (ridge planting, RP; e uniform row planting, UR) e dois níveis de aplicação de nitrogênio do trigo de inverno anterior (N1, 112,50 kg ha-1; N2, 225,00 kg ha-1). Os resultados indicaram que a aplicação de nitrogênio na cultura anterior e no plantio RP da cultura seguinte teve efeitos significativos nas características fotossintéticas e na produtividade do milho verão. Comparado com o plantio UR, este estudo concluiu que RP aumentou o índice de conteúdo de clorofila (CCI), índice de área foliar (LAI), taxa fotossintética líquida (Pn), matéria seca (DM), produtividade e RUE de grãos em 4,1%, 6,4%, 5,2%, 6,4%, 8,9% e 9,4%, respectivamente. Os valores de CCI, LAI, Pn, produtividade e RUE de N2 foram 9,7%, 3,3%, 3,7%, 10,0% e 10,1% superiores aos de N1, respectivamente. RP combinada com a aplicação de nitrogênio na safra anterior de trigo de inverno poderia aumentar os valores de CCI, LAI, Pn, DM, aumentando o rendimento de grãos e RUE do milho do verão seguinte. Concluiu-se que a aplicação prévia de nitrogênio na colheita e o tratamento com padrão RP resultaram em condições ótimas de cultivo para a planície do norte da China.

Four trace elements in pregnant women and their relationships with adverse pregnancy outcomes.

OBJECTIVE: Lack of trace elements during pregnancy is detrimental to maternal and fetal health. Our aim is to study the changes in trace element levels in Chinese pregnant women and their association with adverse pregnancy outcomes. PATIENTS AND METHODS: 1568 cases of Chinese pregnant women in remote areas were collected for a prospective cohort study. Serum copper, zinc, calcium and iron levels were measured at pre-pregnancy, 1st trimester (7w-12w), 2nd trimester (24w-28w) and 3rd trimester (35w-40w). RESULTS: (1) Serum copper levels was significantly higher after pregnancy than before, calcium and iron levels decreased, but zinc levels did not change significantly. (2) Copper and zinc deficiency in pregnant women was not a common finding, but lack of iron and calcium was frequently encountered; iron deficiency was especially common in the 3rd trimester (42.27%). (3) Serum zinc and iron levels in patients who either had a miscarriage or a preterm delivery were significantly lower than in the control group (p < 0.05). In patients with premature rupture of membranes, serum zinc levels were significantly lower (p < 0.05). In patients with intrauterine growth restriction (IUGR), serum copper, zinc, calcium and iron were significantly lower (p < 0.05). CONCLUSIONS: Trace elements is closely associated with fetal growth and development during pregnancy. Deficiency can lead to adverse pregnancy outcomes. Therefore, we should have a reasonable diet, replenish trace elements, therefore reducing the occurrence of adverse pregnancy outcomes.

Leucine-rich repeat-containing G-protein-coupled receptor 8 in the rat brain: Enrichment in thalamic neurons and their efferent projections.

Leucine-rich repeat-containing G-protein-coupled receptor 8 (LGR8; also classified as relaxin family peptide 2 receptor; RXFP2) has been identified as a cognate receptor for the peptide hormone, insulin-like peptide 3 (INSL3) and INSL3-LGR8 signaling plays an essential role in testis descent and germ cell development in human and rodents. Lgr8 mRNA has been detected in human tissues including testis, kidney and brain, but its regional and cellular distribution in these tissues in human or other species is largely unknown. In an initial step to elucidate the physiological function of a putative INSL3-LGR8 system in rat brain, the localization of Lgr8 mRNA was investigated using in situ hybridization histochemistry, revealing a discrete distribution in forebrain, with expression highly enriched in the thalamus. High densities were detected in the parafascicular nucleus (Pf), the dorsolateral, ventrolateral and posterior thalamic nuclei, and in the medial habenula. Lgr8 transcripts were also detected in frontal and motor cortices. The comparative distribution of LGR8 (receptor protein) was examined by autoradiography of [125I]-human INSL3 binding sites, with high densities detected in the thalamus, especially in Pf, and in the entire striatum--the caudate putamen (CPmicro), islands of Calleja, olfactory tubercle, nucleus accumbens--with lower levels in distinct layers of cerebral cortex. Notably, these areas also receive dopaminergic projections. These findings demonstrate the existence of LGR8 in neuronal soma in the thalamus and axons/terminals in thalamic target areas such as the striatum and frontal cortex. LGR8 was also detected throughout the medial habenula-fasciculus retroflexus-interpeduncular nucleus pathway, further indicating that the receptor is transported from mRNA-expressing soma to remote axonal/terminal sites. These findings suggest the existence of a broadly distributed LGR8 signaling system in the rat involved in sensorimotor, limbic and cognitive functions. Further studies are now required to elucidate the precise function of LGR8, under normal and pathological conditions, as importantly, several of the equivalent receptor-positive areas in human brain are part of the pathology of neurodegenerative conditions including Parkinson's disease.

Relaxin-3 in GABA projection neurons of nucleus incertus suggests widespread influence on forebrain circuits via G-protein-coupled receptor-135 in the rat.

Relaxin-3 (RLX3) is a newly identified member of the relaxin/insulin peptide family that is highly conserved across a range of species from fish to mammals and is highly expressed in rat, mouse and human brain. Extensive pharmacological studies have demonstrated that RLX3 is a high affinity, selective ligand for G-protein-coupled receptor-135 (GPCR135, now classified as relaxin family peptide-3 receptor; RXFP3). In ongoing studies to understand the physiological functions of RLX3, the distribution of RLX3-containing neuronal elements in rat brain was determined by immunohistochemistry, using an affinity-purified polyclonal antiserum raised against a conserved segment of the RLX3 C-peptide (AS-R3(85-101)). Consistent with the distribution of RLX3 mRNA, neurons containing RLX3-like immunoreactivity (LI) were observed in the pontine nucleus incertus and the majority of these cells, which are known to express corticotropin-releasing factor receptor-1, were shown to express glutamic acid decarboxylase-65-immunoreactivity, suggesting a GABA phenotype. Nerve fibers and terminals containing RLX3-LI were observed adjacent to cells in the nucleus incertus and in various forebrain regions known to receive afferents from the nucleus incertus, including cortex, septum, hippocampus, thalamus, hypothalamus and midbrain. Regions that contained highest densities of RLX3-positive fibers included the medial septum, lateral preoptic area, lateral hypothalamus/medial forebrain bundle and ventral hippocampus; and additional fibers were observed in olfactory bulb and olfactory and frontal/cingulate cortices, bed nucleus of the stria terminalis, dorsal endopiriform, intergeniculate, and supramammillary nuclei, and the periaqueductal gray and dorsal raphe. The RLX3-positive network overlapped the regional distribution of GPCR135 mRNA and specific binding sites for an [125I]-GPCR135-selective, chimeric peptide. These anatomical findings further support the proposition that RLX3 is the endogenous ligand for GPCR135 in rat brain and provide evidence for broad modulatory activity of RLX3 in behavioral activation relating to autonomic and neuroendocrine control of metabolism and reproduction and higher-order processes such as stress and cognition.

Comparative localization of leucine-rich repeat-containing G-protein-coupled receptor-7 (RXFP1) mRNA and [33P]-relaxin binding sites in rat brain: restricted somatic co-expression a clue to relaxin action?

Relaxin is a polypeptide hormone with established actions associated with reproductive physiology, but until recently the precise nature of the relaxin receptor and its transmembrane signaling mechanisms had remained elusive. In 2002 however, the leucine-rich-repeat-containing G-protein-coupled receptor-7 (now classified as RXFP1) was identified as a cognate receptor for relaxin, with activation resulting in stimulation of intracellular cAMP production. These findings, along with the presence and putative actions of relaxin within the CNS and earlier descriptions of relaxin binding sites in brain, suggest the importance and feasibility of determining if these relaxin binding sites represent leucine-rich-repeat-containing G-protein-coupled receptor-7 and their precise comparative distribution. Thus, the current study reports the distribution of leucine-rich-repeat-containing G-protein-coupled receptor-7 mRNA throughout the rat brain using in situ hybridization histochemistry of [(35)S]-labeled oligonucleotides and the comparative distribution of [(33)P]-human relaxin binding sites. The extensive, topographical distribution of leucine-rich-repeat-containing G-protein-coupled receptor-7 mRNA throughout the adult rat brain correlated very closely to that of [(33)P]-relaxin binding sites. Leucine-rich-repeat-containing G-protein-coupled receptor-7 mRNA was expressed by neurons in several brain regions, including the olfactory bulb, cerebral cortex, thalamus, hippocampus, hypothalamus, midbrain, pons and medulla. Receptor transcripts were most abundant in areas such as the basolateral amygdala, subiculum, deep layers of the cingulate, somatosensory and motor cortices and intralaminar/midline thalamic nuclei. These areas also contained very high densities of [(33)P]-relaxin binding sites, suggesting a largely somatic localization of leucine-rich-repeat-containing G-protein-coupled receptor-7 protein and site of action for relaxin peptide. The central distribution of relaxin-producing neurons has been described, while data on the topography of nerve terminals that contain and secrete the peptide are currently lacking; but overall these findings strongly suggest that leucine-rich-repeat-containing G-protein-coupled receptor-7 is the cognate receptor for relaxin in the rat brain, and support a role for relaxin-leucine-rich-repeat-containing G-protein-coupled receptor-7 signaling in various somatosensory, autonomic and neurohumoral pathways, which warrants further investigation.

Leucine-rich repeat-containing G-protein-coupled receptor 8 in mature glomeruli of developing and adult rat kidney and inhibition by insulin-like peptide-3 of glomerular cell proliferation.

Leucine-rich repeat-containing G-protein-coupled receptor 8 (LGR8, or RXFP2) is a member of the type C leucine-rich repeat-containing G protein-coupled receptor family, and its endogenous ligand is insulin-like peptide-3 (INSL3). Although LGR8 expression has been demonstrated in various human tissues, including testis, ovary, brain and kidney, the precise roles of this receptor in many of these tissues are unknown. In an effort to better understand INSL3-LGR8 systems in the rat, we cloned the full-length Lgr8 cDNA and investigated the presence and cellular localization of Lgr8 mRNA expression in adult and developing rat kidney. On the basis of these findings, we investigated the presence and distribution of renal 125I-labelled human INSL3-binding sites and the nature of INSL3-LGR8 signalling in cultured renal cells. Thus, using in situ hybridization histochemistry, cells expressing Lgr8 mRNA were observed in glomeruli of renal cortex from adult rats and were tentatively identified as mesangial cells. Quantitative, real-time PCR analysis of the developmental profile of Lgr8 mRNA expression in kidney revealed highest relative levels at late stage gestation (embryonic day 18), with a sharp decrease after birth and lowest levels in the adult. During development, silver grains associated with Lgr8 mRNA hybridization were observed overlying putative mesangial cells in mature glomeruli, with little or no signal associated with less-mature glomeruli. In adult and developing kidney, specific 125I-INSL3-binding sites were associated with glomeruli throughout the renal cortex. In primary cultures of glomerular cells, synthetic human INSL3 specifically and dose-dependently inhibited cell proliferation over a 48 h period, further suggesting the presence of functional LGR8 (receptors) on these cells (mesangial and others). These findings suggest INSL3-LGR8 signalling may be involved in the genesis and/or developmental maturation of renal glomeruli and possibly in regulating mesangial cell density in adult rat kidney.

Characterization of the rat INSL3 receptor.

Human LGR8, initially discovered as a low-affinity relaxin receptor, has now been characterized as the INSL3 receptor. To investigate LGR8 function in the rat, an LGR8 ortholog was identified in the rat genome, and the full-length sequence was cloned and expressed. Rat LGR8 bound INSL3 with high affinity, clearly demonstrating that it is the rat INSL3 receptor. Interestingly, native rat relaxin did not activate rat LGR8, indicating that relaxin is not an endogenous ligand for rat LGR8. LGR8 mRNA expression was demonstrated in the gubernaculum at the time of testis descent and in the testis associated with germ cells.

Induction of galanin receptor-1 (GalR1) expression in external granule cell layer of post-natal mouse cerebellum.

Galanin is a modulator of fast transmission in adult brain and recent evidence suggests that it also acts as a trophic factor during neurogenesis and neural injury and repair. Previous studies in our laboratory have identified galanin mRNA in Purkinje cells of adult and developing rat (but not adult mouse) cerebellum; and galanin-binding sites in adult mouse (but not rat) cerebellum. The post-natal development of the cerebellum provides a unique and convenient model for the investigation of developmental processes and to learn more about putative cerebellar galanin systems, the current study examined the presence and distribution of galanin-like-immunoreactivity (- LI), [(125)I]-galanin binding sites and galanin receptor-1 (GalR1) mRNA in post-natal mouse cerebellum. Using autoradiography and in situ hybridization, [(125)I]-galanin binding sites and GalR1 mRNA were first detected on post-natal day 10 (P10) in the external germinal layer of all lobes and high levels were maintained until P14. Quantitative real-time PCR assays detected GalR1 mRNA in whole cerebellum across the post-natal period, with a strong induction and peak of expression at P10. Assessment of galanin levels in whole cerebellum by radioimmunoassay revealed relatively similar concentrations from P5 to P20 and in adult mice (80-170 pg/mg protein), with a significantly higher concentration (250 pg/mg, p < 0.01) detected at P3. These concentrations were some four- to six-fold lower than those in adult forebrain samples. Using immunohistochemistry, galanin-like-immuno-reactivity was observed in prominent fibrous elements within the white matter tracts of the cerebellum at P3-5 and in more punctate elements in the internal granule cell layer and associated with the Purkinje cell layer at P12 and P20. Increased levels of GalR1 mRNA and galanin binding (attributed to GalR1) in the external granule cell layer at P10-12/(14) coincide with granule cell migration from the external to the inner granule cell layer and together with demonstrated effects of other neuropeptide-receptor systems suggest a role for GalR1 signalling in regulating this or related developmental processes.

Atrial natriuretic peptide expression is increased in rat cerebral cortex following spreading depression: possible contribution to sd-induced neuroprotection.

Cortical spreading depression (CSD) is characterised by slowly propagating waves of cellular depolarization and depression and involves transient changes in blood flow, ion balance and metabolism. In cerebral ischaemia, peri-infarct CSD-like depolarization potentiates infarct growth, whereas preconditioning with a CSD episode protects against subsequent ischaemic insult. Thus, many of the long-lasting molecular changes that occur in CSD-affected tissue are presumed to be part of a 'neuroprotective cascade.' 3',5'-Cyclic guanosine monophosphate (cGMP) has been shown to be a neuroprotective mediator and the nitric oxide system, which increases cGMP production by soluble guanylate cyclase, is up-regulated by CSD. Atrial and C-type natriuretic peptide (ANP/CNP) are present in cerebral cortex and their actions are mediated via particulate guanylate cyclase receptors and cGMP production. Therefore, in further efforts to characterise the role of cGMP-related systems in CSD and neuroprotection, this study investigated possible changes in cortical natriuretic peptide expression following acute, unilateral CSD in rats. Using in situ hybridisation, significant 20-80% increases in ANP mRNA were detected in layers II and VI of ipsilateral cortex at 6 h and 1-14 days after CSD. Ipsilateral cortical levels were again equivalent to control contralateral values after 28 days. Assessment of cortical concentrations of ANP immunoreactivity by radioimmunoassay revealed a significant 57% increase at 7 days after CSD. Despite using a sensitive signal-amplification protocol, authentic ANP-like immunostaining was readily detected in subcortical nerve fibres, but was not reliably detected in normal or CSD-affected neocortex, suggesting the presence of very low levels, and/or active or differential processing of the peptide. Cortical CNP mRNA levels are not altered by CSD, indicating the specificity of the observed effects.Overall, these novel findings demonstrate a prolonged increase in cortical ANP expression after an acute episode of CSD. The overlap between the described time course of CSD-induced protection against ischaemic insult and demonstrated increases in ANP levels, suggest that ANP (like nitric oxide) may contribute to CSD-induced neuroprotection, via effects on cGMP production and other signal-transduction pathways.

Differential modulatory effects of alpha- and beta-adrenoceptor agonists and antagonists on cortical immediate-early gene expression following focal cerebrocortical lesion-induced spreading depression.

Unilateral, focal cerebrocortical lesion (FCL) and associated spreading depression (SD) increase immediate-early gene (IEG) expression throughout the ipsilateral hemisphere. Noradrenergic transmission is involved in the regulation of basal- and stimulation-induced expression of IEGs in cerebral cortex; and is modulated by both injury and SD. The present study further investigated the association between the noradrenergic system and cortical adaptive responses, by examining basal and FCL(SD)-induced cortical IEG expression following acute treatment with alpha(1)-, alpha(2)- and beta(1/2)-adrenoceptor (AR) agonists or antagonists. Activation of alpha(1)-ARs by NVI-085, or beta-ARs by salbutamol, increased cortical NGFI-A, c-jun and c-fos mRNA levels, whereas inhibition of alpha(1)-ARs by prazosin, or beta-ARs by propranolol, had no marked effect. The alpha(2)-AR agonists, clonidine and UK14304 also had no effect on basal IEG levels, while blockade of alpha(2)-ARs by methoxyidazoxan significantly increased NGFI-A and c-fos expression, but decreased c-jun mRNA levels. This latter effect confirms the complex and differential nature of IEG regulation in brain. In FCL(SD) rats, all AR agonists generally produced a supra-additive (synergistic) effect on expression of the examined IEGs, compared with drug-treatment or FCL alone. Prazosin reduced FCL(SD)-induced elevations of c-jun and c-fos, but not NGFI-A, mRNA. Methoxyidazoxan enhanced NGFI-A and c-fos mRNA expression after FCL(SD), but reduced c-jun. Propranolol enhanced all lesion-induced IEG levels. These results confirm that alpha(1)- and beta-ARs normally mediate a stimulatory, and alpha(2)-ARs a net inhibitory, influence on cortical cell activity (reflected by NGFI-A, c-fos expression); and demonstrate that alterations in noradrenergic tone modulate the level of cellular activation during and after SD, which is primarily elicited by K(+)/glutamate via NMDA receptors and Ca(2+)-associated mechanisms. In turn, noradrenergic transmission and interactions with excitatory systems are likely to be important in responses to brain injury, including regulation of IEGs and their downstream target genes.

Prolonged induction of neuronal NOS expression and activity following cortical spreading depression (SD): implications for SD- and NO-mediated neuroprotection.

Cortical spreading depression (CSD) is associated with various short- and long-term physiological and neurochemical changes and has been shown to confer an increased susceptibility to accompanying ischemic injury or provide protection against a subsequent experimental ischemia. Nitric oxide is involved in the processes of ischemic injury and under certain conditions mediates cellular protection. To investigate the possibility that CSD-induced alterations in nitric oxide synthase (NOS) expression and activity occur and might be associated with the time-dependent enhancement or prevention by CSD of ischemic damage, this study examined the spatiotemporal changes in nNOS expression and activity in cerebral cortex following CSD. Anesthetized rats had unilateral CSD induced by a 10-min topical application of KCl and were killed at various times thereafter. CSD increased both nNOS mRNA and protein levels throughout layers II-III of cortex. nNOS mRNA in the affected neocortex was significantly increased by 30-90% at 2, 7, and 14 days (P < or = 0.05) compared with contralateral levels, but was not significantly above control values at 1-6 h, 1 day, and 28 days after CSD induction. Levels of [3H]-L-N(G)-nitroarginine binding to NOS were increased by 40-170% 7, 14, and 28 days (P < or = 0.01) after CSD in a similar, but delayed, profile to nNOS mRNA. Levels of nNOS-immunoreactivity were also increased in both neurons and astrocytes of ipsilateral cortex 7 and 14 days after CSD--confirmed by double-immunofluorescence localization. Ex vivo NOS activity in layers I-III of ipsilateral cortex was also increased by 30-50% (P < or = 0.01) at 7 and 14 days after CSD, times coincident with reported maximal ischemic protection. These results demonstrate that nNOS is up-regulated by cellular depolarization/depression occurring during CSD, or by resultant stimuli and suggest that "CSD-conditioned" cortex may be capable of producing appropriate levels of NO to mediate or contribute to protective/adaptive responses to subsequent physical ischemic injury.

Increased striatal proenkephalin mRNA subsequent to production of spreading depression in rat cerebral cortex: activation of corticostriatal pathways?

Cortical Spreading Depression (CSD) is a slowly propagating wave of depolarization and negative interstitial DC potential, that when induced in the rat brain extends across the entire homolateral hemisphere. Despite evidence that CSD does not penetrate into subcortical regions, neurochemical changes in areas anatomically connected to cortex have been reported. In this study in situ hybridization histochemistry was used to examine the levels of cholecystokinin (CCK), proenkephalin (ENK) and prodynorphin (DYN) mRNA in cortex and forebrain basal ganglia following KCl-induced CSD. Unilateral CSD was induced by topical application of 3 M KCl ( approximately 10 microliter) onto the right parietal cortex for 10 min and rats were then killed 1-6 h and 1-28 days later. CCK mRNA levels were increased (P<0.01) in the ipsilateral neocortex 3 h after CSD (13% above levels in contralateral side), reached a peak at 2 days ( approximately 70%) and were still elevated at 7 (30%) but not, 14 or 28 days later. Unilateral CSD also produced a rapid and sustained increase (P<0.05) in ENK mRNA in ipsilateral piriform cortex (from 3 h to 2 days; 70-250% above contralateral), and a delayed increase in caudate putamen and olfactory tubercle at 1 and 2 days ( approximately 25% in both regions), but levels were again equivalent to control at 7 days and beyond. In contrast, no marked changes in neocortical ENK mRNA, or DYN mRNA in both cortex and basal ganglia, were observed under these conditions. These findings demonstrate that CSD has specific, rapid and long-lasting effects on neuropeptide expression in neocortex and subcortical areas. CSD-induced changes in mesostriatal ENK mRNA are proposed to reflect synaptic activation of local neurons via cortical afferent projections.

Differential increases in chromogranins, but not synapsin I, in cortical neurons following spreading depression: implications for functional roles and transmitter peptide release.

Experimental damage of cerebral cortex induces a slow-moving depolarization and subsequent depression of activity called cortical spreading depression (CSD) which is associated with various ionic, metabolic and genomic changes. Chromogranins are a family of water-soluble acidic proteins with a widespread distribution in secretory, large dense-core vesicles of neurons. We have earlier reported that secretogranin II (SgII) mRNA is increased in cerebral cortex hours after a unilateral craniotomy which would have induced CSD. To investigate further the regulation of chromogranin systems and the nature of genomic and biochemical changes produced by CSD, this study examined the temporal changes in chromogranin A (CgA), chromogranin B (CgB) and SgII mRNAs and CgB and SgII immunoreactivity (IR) in cerebral cortex and hippocampus following unilateral KCl-induced CSD. For comparison, the levels of mRNA for synapsin I, a protein present in small synaptic vesicles was also examined. Rats were killed at various times after 10 min or 2 h of CSD and levels of chromogranins mRNAs were determined by semiquantitative in situ hybridization histochemistry, while changes in corresponding peptide products were detected by immunohistochemistry. CSD increased both SgII and CgB mRNA levels in ipsilateral cortex--levels of SgII mRNA were significantly (P < 0.01) increased at 1-6 h after CSD (165-225% of levels in contralateral cortex), but were not significantly above control values at later time points. Increased expression of CgB mRNA was delayed and prolonged compared with SgII and was significantly (P < 0.05) increased between 3 and 24 h (120-145%) after CSD, peaked at 2 days (180%), and was still elevated at 1 week (130%) compared with contralateral cortex. No alteration in CgA mRNA was observed in the ipsilateral cortex of the same animals across the entire time-course except for an increase in piriform cortex at 1-2 days. In contrast, levels of synapsin I mRNA in affected cortex were identical to those in contralateral cortex and cortex in sham-operated rats, at all times after CSD. Levels of chromogranin (SN-IR and PE-11-IR) were also increased in ipsilateral cortex following CSD. A strong increase in SN-IR in neuronal cell bodies and fibres was observed at 12 h and a moderate increase in PE-11-IR was observed 24-72 h after CSD. These results demonstrate that chromogranin transcripts and gene products are differentially regulated by neuronal depolarization/depression occurring during CSD and suggest that these chromogranin proteins may have differing functional roles in peptide transmitter release and distinct effects on neuronal function in rat brain.

Differential spatiotemporal alterations in adrenoceptor mRNAs and binding sites in cerebral cortex following spreading depression: selective and prolonged up-regulation of alpha1B-adrenoceptors.

Noradrenaline, an important transmitter in the CNS, is involved in cerebral plasticity and functional recovery after injury. Experimental brain injury, including KCl application onto the brain surface, induces a slow-moving cortical depolarization/depression wave called cortical spreading depression (CSD). Interestingly, CSD does not produce neuronal damage but can protect cortical neurons against subsequent neurotoxic insults, although the mechanisms involved are unknown. This study examined the status of alpha- and beta-adrenoceptors (ARs) in cerebral cortex following CSD. Anesthetized rats had unilateral CSD induced by a 10-min topical application of KCl to the frontoparietal cortex and were killed at various times thereafter. Levels of alpha1-, alpha2-, beta1-, and beta2-AR mRNA and binding were examined using in situ hybridization histochemistry and radioligand autoradiography. Levels of alpha1b-AR mRNA in the affected neocortex were significantly increased by 20-40% at 1, 2, and 7 days (P

Angiotensinogen and natriuretic peptide mRNAs in rat brain: localization and differential regulation by adrenal steroids in hypothalamus.

Adrenal steroids have been shown to modulate angiotensin II and natriuretic peptide systems--peptide synthesis and metabolism--in vitro. In the present study the effects of adrenal steroids on mRNA encoding the angiotensin II precursor, angiotensinogen (AOGEN), and the natriuretic peptides, atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) in the rat hypothalamus were investigated using quantitative in situ hybridization histochemistry of [35S]- and [33P]-labeled oligonucleotide probes. Adrenalectomy produced an apparent overall decrease in preproAOGEN (ppAOGEN) mRNA in presumed astrocytes in the anterior hypothalamus with significant decreases (ANOVA) measured in the medial preoptic area, the ventral region of the medical preoptic area, the paraventricular, suprachiasmatic, supraoptic, and periventricular nuclei. ppAOGEN mRNA levels were restored by both glucocorticoid (dexamethasone; 2 micrograms/ml in drinking water) and mineralocorticoid (aldosterone; 50 micrograms/kg, SC) replacement. Treatment of intact animals with dexamethasone (2 micrograms/ml in drinking water for 5 days) and aldosterone (100 micrograms/kg, SC, daily for 10 days) produced a significant increase in ppAOGEN mRNA in those hypothalamic regions affected by adrenalectomy. ppANP and ppCNP mRNA-positive neurons were successfully detected using [35S]- and [33P]-labeled probes, respectively, and were abundant in the anterior hypothalamus, particularly in the anteromedial preoptic nucleus of the medial preoptic area. In contrast to the effects on ppAOGEN mRNA, however, alterations in adrenal steroid levels did not significantly change ppANP or ppCNP mRNA levels in neurons of the anteromedial preoptic nucleus or in the arcuate nucleus. These results indicate that adrenal steroids modulate AOGEN gene transcription in vivo, consistent with previous reports of increased levels of ppAOGEN mRNA in a number of brain regions in response to acute dexamethasone treatment and reports of decreased AOGEN immunoreactivity in brain regions of adrenalectomized rats. In contrast, despite reports of modulation of hypothalamic ANP immunoreactivity following adrenalectomy and dexamethasone treatment, it would appear that adrenal steroids do not alter the transcription or stability of hypothalamic natriuretic peptides mRNA in vivo.

Chromogranin mRNA levels in the brain as a marker for acute and chronic changes in neuronal activity: effect of treatments including seizures, osmotic stimulation and axotomy in the rat.

Chromogranin/secretogranins are a family of acidic, soluble proteins with a widespread distribution in secretory vesicles of endocrine and nervous tissues. The effects of experimental stimuli of differing duration and intensity on chromogranin B and secretogranin II mRNA levels in relevant areas of the rat brain were examined by in situ hybridization histochemistry using 35S-labelled oligonucleotides. Effects of two 'chronic stimulation' paradigms were studied - the effect of 4 days of water or food deprivation on mRNA levels in the hypothalamus and the effect of unilateral cervical vagotomy on transcript levels in the dorsal vagal complex 1, 2 and 7 days after surgery. After 4 days of water deprivation secretogranin II mRNA was significantly increased in supraoptic nucleus (366 +/- 21% of control, P < 0.01), the magnocellular paraventricular nucleus (209 +/- 20% of control, P < 0.01) and the parvocellular paraventricular nucleus (147 +/- 6% of control, P < 0. 05) after 4 days of food deprivation. Seven days after unilateral cervical vagotomy, secretogranin II and chromogranin B mRNA levels were markedly decreased in the ipsilateral dorsal motor nucleus of the vagus (25 +/- 4 and 47 +/- 8% of contralateral values respectively, P < 0.01). Rapid changes in chromogranin mRNA were also detected following shorter duration 'acute stimulation' - in the hypothalamus after hypertonic saline injection, in the hippocampus after electrical stimulation-induced kindled seizures, and in the cerebral cortex after unilateral craniotomy. A large increase in secretogranin II mRNA was detected in the supraoptic nucleus (202 +/- 25% of control, P < 0.01) and the magnocellular paraventricular nucleus (168 +/- 29% of control, P < 0.05) 3 h after a single intraperitoneal injection of hypertonic (1.8 M) saline. Markedly increased levels of secretogranin II (125-160% of control) and chromogranin B (140-230% of control) mRNA were observed in granule cells of the dentate gyrus 0.5-2 h after amygdaloid stimulation-induced seizures. A moderate increase in secretogranin II mRNA (144 +/- 11% of contralateral side, P < 0.01) was found in the underlying cerebral cortex 2.5 h after unilateral craniotomy. These results indicate that measurement of changes in chromogranin mRNA, particularly secretogranin II, is a useful means of assessing both rapid and long-lasting increases and decreases in neuronal activity and, in contrast to immediate early gene mRNA levels, may better reflect specific changes in neuronal secretory activity associated with transmitter/peptide release.

Effects of ovarian steroids on hypothalamic opioid receptor subtypes in ovariectomized ewes: regional changes in density and affinity.

The negative feedback regulation by ovarian steroids of luteinizing hormone secretion may be partially mediated by a hypothalamic endogenous opioid mechanism. This could be affected by ovarian steroid-regulated changes in hypothalamic opioid receptor binding mechanisms. In this report we show that in the presence of blocking concentrations of site-selective opioid analogues, [3H] diprenorphin homogeneously labelled mu, delta or kappa receptor subtypes respectively. Using this receptor binding model, we characterized each opioid receptor subtype in the hypothalamic preoptic area and medio-basal hypothalamus of ovariectomized (OVX) and OVX plus progesterone- or oestradiol-17 beta (OE2)-treated ewes. In the preoptic area, progesterone treatment did not influence the affinity or capacity of delta or kappa receptor binding sites, but significantly reduced mu receptor subtype content (20% less than control) with no statistically significant change in affinity. There was no effect of OE2 on either the affinity or capacity of each opioid receptor subtype in this area. In the mediobasal hypothalamus, progesterone treatment significantly decreased delta subtype receptor affinity (22 +/- 11 nM vs control 7 +/- 2 nM) and increased binding capacity (78 +/- 9 fmol/mg protein vs control 37 +/- 16 fmol/mg protein). OE2 treatment had a similar, though more profound effect on affinity (51 +/- 17 nM) and binding capacity (139 +/- 26 fmol/mg protein) at the delta receptor binding site. There were no significant changes in the affinity or capacity of mu or kappa binding sites in the medio-basal hypothalamus. These results indicate that steroid hormones modulate hypothalamic opioid receptors in the OVX ewe in a receptor subtype- and region-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Noradrenergic regulation of immediate early gene expression in rat forebrain: differential effects of alpha 1- and alpha 2-adrenoceptor drugs.

Noradrenergic (NAergic) transmission in the rat cerebral cortex has recently been shown to be involved in the regulation of the basal expression of NGFI-A, an immediate early gene (IEG) which encodes a zinc-finger transcription factor. The present study further investigated the role of the NAergic system in mediating cortical IEG expression and possible topographical changes in expression of NGFI-A mRNA in rat forebrain after alpha 1- and alpha 2-adrenoceptor (AR) agonist and antagonist treatment. Expression of c-fos and c-jun, which encode leucine-zipper class transcription factors, was also studied. Male Sprague-Dawley rats were injected intraperitoneally with either an alpha 1-AR agonist (methoxamine, 5 or 10 mg/kg); an alpha 1-AR antagonist (prazosin, 5 mg/kg); an alpha 2-AR agonist (clonidine, 0.5 mg/kg); or an alpha 2-AR antagonist (methoxyidazoxan, 5 mg/kg) and killed after 1 h. IEG mRNA levels were detected by quantitative in situ hybridization histochemistry using 35S-labelled oligonucleotides. High basal levels of NGFI-A mRNA were present in cortical layers IV and VI, hippocampal CA1, piriform cortex, amygdala and caudate putamen. alpha 1-AR agonist and antagonist treatment had essentially no effect on IEG mRNA, despite producing characteristic behavioral and peripheral effects at the doses used. Methoxyidazoxan significantly increased (mean%) NGFI-A mRNA in: cerebral cortex (44); caudate putamen (82); amygdala (92); and CA1 of hippocampus (48), while clonidine significantly decreased NGFI-A mRNA in the various cortical layers to a similar extent (27-37%). Basal c-fos mRNA expression was lower than that for NGFI-A in forebrain areas including cortex, caudate putamen and hippocampus.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of immunoreactive AVP in the ovine hypothalamo-pituitary axis.

The aims of these studies were to determine the precise molecular nature of immunoreactive (IR-) vasopressin (AVP) in the ovine hypothalamo-pituitary axis and to examine a possible role for glucocorticoids in regulating both AVP processing and levels in this axis. The IR-AVP in extracts of paraventricular nucleus, median eminence, portal blood, and anterior and neurointermediate pituitary elutes as a single peak on two distinct HPLC solvent systems, suggesting that AVP is processed identically in these tissues. Identical profiles were also found in extracts from pituitaries and sheep subjected to chronic (10 days) glucocorticoid treatment, or hypothalamo-pituitary disconnection. The latter findings confirm that in sheep, like the rat, AVP is synthesized and processed in the anterior pituitary and is not sequestered from extrapituitary sources.

Morphine decreases LH secretion in ovariectomized ewes only after steroid priming and not by direct pituitary action.

To determine whether opiates directly modulate pituitary LH secretion in vivo, morphine was administered to hypothalamo-pituitary-disconnected (HPD) ewes which were receiving exogenous pulses of GnRH. To define the steroidal background which is permissive to a morphine-induced decrease in LH secretion, ovariectomized (OVX) ewes were treated as follows in groups of four: group 1, no implant; group 2, small 17 beta-estradiol (E2) (1 cm long x 0.33 diameter) and progesterone (P) implants; group 3, medium E2 (1 cm long x 0.46 diameter) and P implants, and group 4, medium E2 implants. Jugular blood samples were taken at 10-min intervals for 9 h, during which there was a 3-hour pretreatment period, a 3-hour treatment period when the sheep were given six intravenous injections of 10 mg morphine every 30 min, and a 3-hour run-off period. Morphine inhibited the mean plasma concentrations of LH and LH pulse frequency in group 3 only, and in 2/4 ewes in this group LH secretion was abolished and did not return to a pulsatile mode during the 3-hour run-off sampling period. In a second experiment designed to test the pituitary action of morphine, OVX-HPD ewes were primed with medium E2 and P implants and were given hourly pulses of 250 ng GnRH intravenously. Jugular blood samples were taken around each GnRH pulse over an 8-hour period. The first three pulses served as a control sampling period, after which the sheep were treated with morphine (six intravenous injections of 10 mg morphine every 30 min).(ABSTRACT TRUNCATED AT 250 WORDS)