Comprehensive analysis of histophysiology, transcriptome and metabolome tolerance mechanisms in black porgy (Acanthopagrus schlegelii) under low temperature stress.

Low temperature stress has adverse effects on fish growth and reproduction, causing huge economic losses to the aquaculture industry. Especially, black porgy (Acanthopagrus schlegelii) farming industry in north of Yangtze River has been severely affected by low temperature for a long time. To explore the tolerance mechanism of black porgy to low temperature stress, the experiment was designed. The liver and gill tissues of black porgy were taken from the water temperature point of 15 °C (control group named as CG), 3.8 °C (cold sensitive group named as CS) and 2.8 °C (cold tolerant group named as CT) with a cooling rate of 3 °C/d from 15 °C for histophysiology, transcriptomics and metabolomics analysis. After cold stress, the histological results showed that the nucleus of the black porgy liver tissue appeared swelling, the cell arrangement was disordered; meanwhile the gill lamellae were twisted and broken, the epidermis was detached and aneurysm appeared. In addition, the expression of antioxidant, glucose metabolism and immune-related enzymes in the liver and gill of black porgy also changed significantly after low temperature stress. By analyzing the transcriptome and metabolome dates of black porgy liver, 3474 differentially expressed genes (DEGs) and 689 differentially expressed metabolites (DEMs) involved in low temperature stress were identified, respectively. The results of the transcriptome and metabolome combined analysis showed that individuals in the CS group mainly supplied energy to the body through lipid metabolism and amino acid metabolism, and meanwhile the apoptosis pathway was activated. While, individuals in the CT group mainly through glucose metabolism and steroid hormone biosynthesis to supply energy for the body. The validation results of qPCR on eight functional genes further demonstrated the reliability of RNA-Seq data. In summary, the results provide molecular information about adaptation to climate change and genetic selection of black porgy.

[Effects of Biochar Application on Soil Organic Carbon Component in Eucalyptus Plantations After Five Years in Northern Guangxi].

To investigate the effects of biochar(BC) addition on soil organic carbon(SOC) contents and its fractions under different biochar applications, Eucalyptus waste twigs in Northern Guangxi were used to produce BC at 500℃. Additionally, we sought to clarify and define the carbon sequestration potential of soil and provide a basis for the preparation of biochar from Eucalyptus forest wastes and soil improvement. In a long-term positioning test of biochar application from 1997, six different treatments were selected:0(CK), 0.5%(T1), 1%(T2), 2%(T3), 4%(T4), and 6%(T5). The contents of SOC, light fraction organic carbon(LFOC), heavy fraction organic carbon(HFOC), easily oxidized organic carbon(EOC), dissolved organic carbon(DOC), particulate organic carbon(POC), microbial biomass carbon(MBC), and carbon stock(CS) following the different treatments were measured. The results showed that:① compared to that in the control, biochar application induced an increase in each soil organic carbon fraction with increasing application rate and reached a maximum under the T4 or T5 treatments; with the increase in biochar application, the contents of SOC, DOC, EOC, POC, MBC, and CS increased significantly by 101.62%, 67.46%, 143.03%, 164.78%, 110.88%, and 41.73%, respectively. ② The contents of LFOC and HFOC in the 0-10, 10-20, and 20-30 cm soil layers increased significantly by 41.41%-140.63%, 9.26%-87.04%, and -19.54%-106.90% and 15.32%-78.99%, 15.72%-75.25%, and 89.49%-148.64%, respectively, with the increase in biochar application. The average contents of LFOC and HFOC in the 0-30 cm soil layer also increased gradually. The soil carbon pool of the Eucalyptus forest was dominated by a relatively stable heavy fraction organic carbon. ③ The contents of carbon stock, soil organic carbon, and its fractions decreased with the increase in soil depth. In conclusion, the application of forestry waste biochar for five years could significantly increase the content of SOC and its components, thereby increasing soil organic carbon activity. Therefore, increasing the amount of biochar was an effective measure to enhance the carbon storage, soil stable carbon pool, and soil quality of the Eucalyptus plantation field. This study provides a reference for the resource utilization of forestry waste and improvements in soil fertility of Eucalyptus plantations.

[Effects of Biochar Application on Soil Organic Nitrogen Component and Active Nitrogen in Eucalyptus Plantations After Five Years in Northern Guangxi].

The objective of this study was to research the characteristics of fractions of organic nitrogen and active nitrogen and their relationship under different biochar applications and to provide a basis for the preparation and practical application of biochar from Eucalyptus forest wastes. In a long-term positioning test of biochar application from 2017, six different treatments were selected:0(CK), 0.5%(T1), 1%(T2), 2%(T3), 4%(T4), and 6%(T5). The contents of soil organic nitrogen components, total nitrogen(TN), dissolved organic nitrogen(DON), and microbial biomass nitrogen(MBN) following the different treatments were measured. The results showed that:① compared with that of the control, with the increase in biochar application, the contents of soil TN, acidolysis of total organic nitrogen(AHON), ammonia nitrogen(AN), amino acid nitrogen(AAN), MBN, DON, and nitrogen storage(NS) increased significantly by 45.48%-156.32%, 44.31%-171.31%, 38.06%-223.37%, 39.42%-163.32%, 36.72%-109%, 23.27%-113.51%, and 29.45%-62.37%, respectively. The contents of soil hydrolyzable unknown nitrogen(HUN) and non-hydrolyzable nitrogen(NHN) also increased significantly by 88.41%-158.71% and 50.24%-139.01%, respectively. The contents of soil amino sugar nitrogen(ASN) decreased by 7.72%-32.73%. The contents of different forms of organic nitrogen fractions in all treatments displayed an order of AN > AAN > NHN > HUN > ASN. Compared with the no biochar treatment, each biochar treatment increased the contents and proportion of AHON in the TN. ② With the exception of HUN, the contents of other soil organic nitrogen components and active nitrogen content decreased with the increase in soil depth. ③ There were significantly positive correlations between TN, MBN, and DON and AHON, NHN, and NS contents. The principal component analysis showed that bulk density and ASN and TN and HUN, AAN, DON, and AHON were closely related, respectively. In conclusion, the application of forestry waste biochar for five years could significantly increase the content of soil organic nitrogen component and active nitrogen, thereby improving the capacity of the soil to supply nitrogen. AHON, AN, and AAN were the main factors contributing to soil active nitrogen content.

Control of the Structural Charge Distribution and Hydration State upon Intercalation of CO2 into Expansive Clay Interlayers.

Numerous experimental investigations indicated that expansive clays such as montmorillonite can intercalate CO2 preferentially into their interlayers and therefore potentially act as a material for CO2 separation, capture, and storage. However, an understanding of the energy-structure relationship during the intercalation of CO2 into clay interlayers remains elusive. Here, we use metadynamics molecular dynamics simulations to elucidate the energy landscape associated with CO2 intercalation. Our free energy calculations indicate that CO2 favorably partitions into nanoconfined water in clay interlayers from a gas phase, leading to an increase in the CO2/H2O ratio in clay interlayers as compared to that in bulk water. CO2 molecules prefer to be located at the centers of charge-neutral hydrophobic siloxane rings, whereas interlayer spaces close to structural charges tend to avoid CO2 intercalation. The structural charge distribution significantly affects the amount of CO2 intercalated in the interlayers. These results provide a mechanistic understanding of CO2 intercalation in clays for CO2 separation, capture, and storage.

Ambient climate determines the directional trend of community stability under warming and grazing.

Changes in ecological processes over time in ambient treatments are often larger than the responses to manipulative treatments in climate change experiments. However, the impacts of human-driven environmental changes on the stability of natural grasslands have been typically assessed by comparing differences between manipulative plots and reference plots. Little is known about whether or how ambient climate regulates the effects of manipulative treatments and their underlying mechanisms. We collected two datasets, one a 36-year long-term observational dataset from 1983 to 2018, and the other a 10-year manipulative asymmetric warming and grazing experiment using infrared heaters with moderate grazing from 2006 to 2015 in an alpine meadow on the Tibetan Plateau. The 36-year observational dataset shows that there was a nonlinear response of community stability to ambient temperature with a positive relationship between them due to an increase in ambient temperature in the first 25 years and then a decrease in ambient temperature thereafter. Warming and grazing decreased community stability with experiment duration through an increase in legume cover and a decrease in species asynchrony, which was due to the decreasing background temperature through time during the 10-year experiment period. Moreover, the temperature sensitivity of community stability was higher under the ambient treatment than under the manipulative treatments. Therefore, our results suggested that ambient climate may control the directional trend of community stability while manipulative treatments may determine the temperature sensitivity of the response of community stability to climate relative to the ambient treatment. Our study emphasizes the importance of the context dependency of the response of community stability to human-driven environmental changes.

[Effects of Eucalyptus Branches Biochar Application on Soil Physicochemical Properties of Acidified Soil in a Eucalyptus plantation in Northern Guangxi].

This study aims to explore the effects of different biochar applications on soil physical and chemical properties in a Eucalyptus plantation in Northern Guangxi, find the best biochar application amount, and provide scientific guidance for the efficient utilization of forest residue and soil improvement. The soil of a four-year Eucalyptus plantation at the Huangmian forest farm in Northern Guangxi was selected as the study area, and six treatments including 0 (CK), 0.5% (T1), 1.0% (T2), 2% (T3), 4% (T4), and 6% (T5) were set through a field-positioning experiment to analyze the changes in soil physical and chemical properties under different application rates. Compared with the 0-30 cm soil layer of the control treatment, biochar application decreased the mean soil bulk by 3.82%-33.55%, while it increased the soil natural water content, capillary porosity, and total capillary porosity by 7.67%-31.75%, 8.95%-33.19%, and 9.28%-35.86%, respectively. The contents of exchangeable acid, exchangeable aluminum, exchangeable hydrogen, and exchangeable sodium in the soil decreased by 8.28%-70.03%, 5.55%-70.34%, 5.10%-21.78%, and 12.81%-49.27%. Biochar application increased the cation exchange capacity, electrical conductivity, exchangeable magnesium, and exchangeable calcium by 27.08%-160.39%, 117.00%-546.64%, 17.10%-66.14%, and 17.38%-71.38%, respectively. Soil pH increased by 0.17-1.29 after biochar addition. Similarly, the contents of soil organic carbon, total phosphorus, total potassium, available nitrogen, available phosphorus, and available potassium increased by 10.94%-51.37%, 14.29%-59.45%, 6.48%-59.57%, 6.28%-29.41%, 4.79%-19.81%, and 7.72%-75.87%. There was a positive correlation among the main physical and chemical factors. The physical and chemical properties reached their maximum values in the T4 or T5 treatment (4% or 6%). Biochar application provided considerable relief from soil acidification in the Eucalyptus plantation and had a positive effect on soil physicochemical properties. The addition 4%-6% of ripe Eucalyptus biochar produced the optimum results. The results show that biochar can improve the physical and chemical properties of soil, increase soil fertility, and enhance the soil's ability to retain water and fertilizer after twelve months. The findings of this study can be used as a reference in practical applications for soil improvement and sustainable management of Eucalyptus plantations.

Effects of natural zeolites on field-scale geologic noble gas transport.

Improving predictive models for noble gas transport through natural materials at the field-scale is an essential component of improving US nuclear monitoring capabilities. Several field-scale experiments with a gas transport component have been conducted at the Nevada National Security Site (Non-Proliferation Experiment, Underground Nuclear Explosion Signatures Experiment). However, the models associated with these experiments have not treated zeolite minerals as gas adsorbing phases. This is significant as zeolites are a common alteration mineral with a high abundance at these field sites and are shown here to significantly fractionate noble gases during field-scale transport. This fractionation and associated retardation can complicate gas transport predictions by reducing the signal-to-noise ratio to the detector (e.g. mass spectrometers or radiation detectors) enough to mask the signal or make the data difficult to interpret. Omitting adsorption-related retardation data of noble gases in predictive gas transport models therefore results in systematic errors in model predictions where zeolites are present.Herein is presented noble gas adsorption data collected on zeolitized and non-zeolitized tuff. Experimental results were obtained using a unique piezometric adsorption system designed and built for this study. Data collected were then related to pure-phase mineral analyses conducted on clinoptilolite, mordenite, and quartz. These results quantify the adsorption capacity of materials present in field-scale systems, enabling the modeling of low-permeability rocks as significant sorption reservoirs vital to bulk transport predictions.

Redefining Pyropia (Bangiales, Rhodophyta): Four New Genera, Resurrection of Porphyrella and Description of Calidia pseudolobata sp. nov. From China.

The cosmopolitan red algal genus Pyropia sensu lato is the most speciose of the bladed Bangiales genera. In a major revision of the Bangiales, Pyropia was resurrected from Porphyra, although there was evidence at the time that species of Pyropia could be separated into several genera. Subsequent global phylogenetic analyses continued to resolve species assigned to Pyropia into several major clades with strong support, and the latest biogeographic analyses indicated that species distribution was also a pointer to the underlying phylogeny of Pyropia sensu lato. Therefore, in the present study, we have redefined the genus Pyropia, resurrected Porphyrella, and proposed four new genera: Calidia, Neoporphyra, Neopyropia, and Uedaea. Based on a molecular phylogenetic study of the bladed Bangiales of China, a species which did not match any known taxa was resolved in the new genus Calidia. The species, Calidia pseudolobata sp. nov., is described based on both morphological and molecular data. Molecular sequence data for rbcL, 18S, and COI-5P were amplified for 15 samples in the present study. All the obtained rbcL sequences were identical to each other except for one (LYCN117) with one base pair difference. Two haplotypes of 18S (V9 region) were observed with one base pair difference (C/T30 ). All the obtained COI-5P sequences were identical. Morphological comparisons were conducted not only with species in Calidia, but also with generically uncertain species currently assigned to Porphyra.

Profiling the Bacterial Diversity in a Typical Karst Tiankeng of China.

While karst tiankengs have a higher capacity to act as safe havens for biodiversity in changing climates, little is known about their soil microorganisms. To fill this gap, we investigate the distribution and driving factors of the bacterial community in karst tiankeng systems. There is a significant difference in the soil characteristics between the inside and the outside of a karst tiankeng. At the karst tiankeng considered in this study, the bacterial composition, in terms of the operational taxonomic unit (OTU), was found to be significantly different in different soil samples, taken from diverse sampling sites within the collapsed doline or the external area, and showed a high habitat heterogeneity. The dominant phylum abundances vary with the sampling sites and have their own indicator taxa from phylum to genus. Unlike the primary controlling factors of plant diversity, the microclimate (soil moisture and temperature), soil pH, and slope dominated the distribution of the bacterial community in karst tiankeng systems. Our results firstly showed the distribution characteristics of bacterial communities and then revealed the importance of microhabitats in predicting the microbial distribution in karst tiankeng systems.

[Soil Organic Carbon Distribution and Components in Different Plant Communities Along a Water Table Gradient in the Huixian Karst Wetland in Guilin].

In order to reveal the effect of vegetation type and soil physicochemical properties on the distribution of soil organic carbon and its components, a field survey was carried out on nine different plant communities along a water table gradient in the Huixian wetland with samples of soil at 0-10 cm, 10-20 cm, and 20-30 cm in depth. The soil organic carbon (SOC), light fraction organic carbon (LFOC), heavy fraction organic carbon (HFOC), easily oxidized organic carbon (EOC), dissolved organic carbon (DOC), particulate organic carbon (POC), and microbial biomass carbon (MBC) were measured. The correlations among soil organic carbon components and soil physicochemical properties were also examined. The results showed that:① The average proportion of LFOC and HFOC to SOC at 0-30 cm soil depth was 11.10% and 88.90%, respectively. The distribution ratio of the heavy component was much higher than of the light component in soils. ② The content of SOC, DOC, EOC, POC, and MBC (except in the Panicum repens community) and the values of DOC/SOC, EOC/SOC, and POC/SOC all decreased with increase of the soil depth. ③ Among the nine different plant communities, the contents of SOC, LFOC, HFOC, MBC, DOC, EOC, and POC of Cladium chinense were significantly higher than for other communities in same soil layers. ④ There were significantly positive correlations among soil organic carbon components (SOC) and soil total nitrogen (TN). LFOC, HFOC, DOC, and POC were also positively correlated with soil pH. The soil bulk density was significantly negative correlated with LFOC, HFOC, DOC, EOC, and POC, and the content of clay was also negatively correlated with LFOC, HFOC, DOC, POC, and MBC. ⑤ Path analysis showed that TN, soil pH, soil sand content, and soil water content (SWC) has indirect effects on HFOC by influencing other soil factors. Soil TN had strong positive effects on EOC, DOC, and POC, and SWC also has the largest direct negative effect on MBC. This showed that there were close interactions between soil physicochemical properties and soil organic carbon components. This study may provide a reference base for sustainable development and scientific predictions regarding the Huixian Karst wetland.

[Effects of Reclamation on Soil Nutrients and Microbial Activities in the Huixian Karst Wetland in Guilin].

To investigate the effect of reclamation on soil quality in the Huixian Karst Wetland, samples from different soil levels were collected from marsh wetland, reclaimed paddy field, and reclaimed dry farmland, for analyzing soil nutrient (carbon, nitrogen, phosphorous, and potassium) contents, microbial biomass carbon/nitrogen (MBC/MBN), and microbial activity indicators[i.e. basal respiration (BR), potential respiration (PR), microbial quotient (qMB), and metabolic quotient (qCO2)]. The correlations between the soil nutrient contents and soil microbial activity indictors were examined. The results showed that:①Artificial reclamation led to the trend of slight acidity in the soil and a marked loss in soil nutrients, while, the pH value, soil water content (SWC), and the contents of soil organic carbon (SOC), total nitrogen (TN), available nitrogen (AN), total phosphorus (TP), available phosphorus (AP), total potassium (TK), and available potassium (AK) decreased with reclamation. ②Among all the microbes, bacteria were the most numerous, followed by actinomycetes, and fungi were the least numerous. The microbial quantity decreased with the increase in the soil depth on the whole. The proportion of bacteria and actinomycetes were much higher in the paddy field, and that of fungi was the highest in the dry farmland. ③ In total, protease, sucrase, urease, catalase, and polyphenol oxidase activities decreased with the increasing of soil depths. Soil reclamation reduced the soil enzyme activities. ④qCO2 decreased after an initial increase in the marsh wetland, while it rose gradually in the reclaimed paddy field and reclaimed dry farmland. The contents of MBC, MBN, BR, PR, and qMB were the highest in the marsh wetland, followed by those in the reclaimed paddy field, with the lowest contents occurring in the reclaimed dry farmland. The trend of qCO2 contents in the 0-10 cm and 10-20 cm soil layers followed the order of marsh wetland > paddy field > dry farmland, but in the 20-30 cm and 30-40 cm soil layers, it showed the order dry farmland > paddy field > marsh wetland. The continuation of reclamation resulted in the decrease in soil microbial activity, and soil quality as well, especially in the dry farmland. Meanwhile, we should reduce the areas of paddy fields and dry farmlands under reclamation during the process of wetland ecological restoration in future. Conversion of farmlands to wetlands or lakes, to improve and increase the size of wetland ecosystems of nearby lands, should be done gradually.

A molecular phylogeny of the bladed Bangiales (Rhodophyta) in China provides insights into biodiversity and biogeography of the genus Pyropia.

A molecular taxonomic study was undertaken for the first time of the bladed Bangiales of the mainland coast of China (Northwest Pacific) based on sequence data of 201 plastid rbcL and 148 nuclear 18S sequences of historical and contemporary specimens. The results revealed that only one genus of bladed Bangiales, Pyropia, was present along Chinese coast. Species delimitation was determined using two empirical methods: the Automatic Barcode Gap Discovery (ABGD) and General Mixed Yule Coalescence (GMYC) coupled with detection of monophyly in tree reconstruction. At least fourteen species of Pyropia were recovered. Six species were confirmed that had been recorded previously based on morphology (Py. suborbiculata, Py. yezoensis, Py. haitanensis, Py. katadae, Py. tenera and Py. acanthophora), three species were recorded from China for the first time (Py. kinositae, Py. pseudolinearis and Py. tanegashimensis), and five cryptic species that did not match any molecular sequences were also discovered. The phylogeny of the concatenated rbcL and 18S dataset resolved three singletons and four clades. Each clades has a strong trend towards occupying a biogeographic region, but they are not confined to them. A transoceanic and antitropical pattern of distribution was found for Pyropia at both the subgeneric and species level. This together with high biodiversity (ca. 30% of all known Pyropia species) indicates that the Northwest Pacific might act as a centre of origin for modern distribution of Pyropia since the early Cenozoic.

Growth and nutrient removal of three macrophytes in response to concentrations and ratios of N and P.

Wastewater from different sources shows great differences in concentrations and ratios of N and P. In order to choose suitable plant species to remove excess N and/or P from polluted waters, it is important to know the performances of these plants under different N and P concentrations. In this study, we investigated the growth and N and P removal rate of three macrophytes, Coix lacryma-jobi, Iris wilsonii, and Arundo donax under six N and P combination treatments. C. lacryma-jobi preferred higher N and P concentrations (16 mg N L-1 and 3.2 mg P L-1), and increasing N supply could increase its P removal rate. I. wilsonii exhibited a growth preference at a combination of moderate N and P concentrations (8 mg N L-1 and 0.8 mg P L-1). A. donax could grow well at all combinations of N and P and had significantly higher relative growth rate and N and P removal rates than the other two species. These results showed A. donax is a promising species to treat various polluted waters and the other two species can be used specifically to treat certain types of wastewater.

Effects of warming and grazing on soil N availability, species composition, and ANPP in an alpine meadow.

Uncertainty about the effects of warming and grazing on soil nitrogen (N) availability, species composition, and aboveground net primary production (ANPP) limits our ability to predict how global carbon sequestration will vary under future warming with grazing in alpine regions. Through a controlled asymmetrical warming (1.2/1.7 degrees C during daytime/nighttime) with a grazing experiment from 2006 to 2010 in an alpine meadow, we found that warming alone and moderate grazing did not significantly affect soil net N mineralization. Although plant species richness significantly decreased by 10% due to warming after 2008, we caution that this may be due to the transient occurrence or disappearance of some rare plant species in all treatments. Warming significantly increased graminoid cover, except in 2009, and legume cover after 2008, but reduced non-legume forb cover in the community. Grazing significantly decreased cover of graminoids and legumes before 2009 but increased forb cover in 2010. Warming significantly increased ANPP regardless of grazing, whereas grazing reduced the response of ANPP to warming. N addition did not affect ANPP in both warming and grazing treatments. Our findings suggest that soil N availability does not determine ANPP under simulated warming and that heavy grazing rather than warming causes degradation of the alpine meadows.

Selective activation of the M1 muscarinic acetylcholine receptor achieved by allosteric potentiation.

The forebrain cholinergic system promotes higher brain function in part by signaling through the M(1) muscarinic acetylcholine receptor (mAChR). During Alzheimer's disease (AD), these cholinergic neurons degenerate, therefore selectively activating M(1) receptors could improve cognitive function in these patients while avoiding unwanted peripheral responses associated with non-selective muscarinic agonists. We describe here benzyl quinolone carboxylic acid (BQCA), a highly selective allosteric potentiator of the M(1) mAChR. BQCA reduces the concentration of ACh required to activate M(1) up to 129-fold with an inflection point value of 845 nM. No potentiation, agonism, or antagonism activity on other mAChRs is observed up to 100 microM. Furthermore studies in M(1)(-/-) mice demonstrates that BQCA requires M(1) to promote inositol phosphate turnover in primary neurons and to increase c-fos and arc RNA expression and ERK phosphorylation in the brain. Radioligand-binding assays, molecular modeling, and site-directed mutagenesis experiments indicate that BQCA acts at an allosteric site involving residues Y179 and W400. BQCA reverses scopolamine-induced memory deficits in contextual fear conditioning, increases blood flow to the cerebral cortex, and increases wakefulness while reducing delta sleep. In contrast to M(1) allosteric agonists, which do not improve memory in scopolamine-challenged mice in contextual fear conditioning, BQCA induces beta-arrestin recruitment to M(1), suggesting a role for this signal transduction mechanism in the cholinergic modulation of memory. In summary, BQCA exploits an allosteric potentiation mechanism to provide selectivity for the M(1) receptor and represents a promising therapeutic strategy for cognitive disorders.

Methane emission by plant communities in an alpine meadow on the Qinghai-Tibetan Plateau: a new experimental study of alpine meadows and oat pasture.

Recently, plant-derived methane (CH(4)) emission has been questioned because limited evidence of the chemical mechanism has been identified to account for the process. We conducted an experiment with four treatments (i.e. winter-grazed, natural alpine meadow; naturally restored alpine meadow eight years after cultivation; oat pasture and bare soil without roots) during the growing seasons of 2007 and 2008 to examine the question of CH(4) emission by plant communities in the alpine meadow. Each treatment consumed CH(4) in closed, opaque chambers in the field, but two types of alpine meadow vegetation reduced CH(4) consumption compared with bare soil, whereas oat pasture increased consumption. This result could imply that meadow vegetation produces CH(4). However, measurements of soil temperature and water content showed significant differences between vegetated and bare soil and appeared to explain differences in CH(4) production between treatments. Our study strongly suggests that the apparent CH(4) production by vegetation, when compared with bare soil in some previous studies, might represent differences in soil temperature and water-filled pore space and not the true vegetation sources of CH(4).

Hypothalamic projections to locus coeruleus neurons in rat brain.

Locus coeruleus (LC) neurons respond to autonomic and visceral stimuli and discharge in parallel with peripheral sympathetic nerves. The present study characterized the synaptic organization of hypothalamic afferents with catecholaminergic neurons in the LC using electron microscopy. Peroxidase labeling of axon terminals that were anterogradely labeled from the paraventricular nucleus (PVN) was combined with gold-silver labeling of tyrosine hydroxylase in the LC. Approximately 19% of the anterogradely labeled axon terminals formed synaptic specializations with tyrosine hydroxylase-immunoreactive dendrites in the LC. Retrograde transport from the LC combined with immunocytochemical detection of enkephalin and corticotropin-releasing factor (CRF) suggested that most of the LC-projecting PVN neurons (30%) were CRF immunoreactive and few (2%) were enkephalin immunoreactive. Finally, dual retrograde tracing from the LC and median eminence revealed that PVN neurons that project to the LC are a population distinct from that projecting to the median eminence. The present data suggest that a population of hypothalamic neurons is poised to directly modulate the activity of LC neurons and may integrate autonomic responses in brain by influencing LC neurons. Moreover, PVN neurons that use CRF as a neurohormone are distinct from those that use CRF as a neuromodulator to impact on the LC.

Chronic morphine sensitizes the brain norepinephrine system to corticotropin-releasing factor and stress.

Chronic opiate use produces persistent changes in brain neurons that are expressed as adverse effects, including physical dependence and compulsive drug-seeking behavior. Dysregulation of the hypothalamic-pituitary-adrenal response to stress also occurs with chronic opiate administration and has been implicated as a contributing factor to continued substance abuse. This study provides the first evidence for dysregulation of the central noradrenergic response to stress by chronic opiates. Chronic morphine selectively sensitized locus ceruleus (LC)-norepinephrine (NE) neurons to corticotropin-releasing factor (CRF), an integral mediator of the stress response. CRF doses that were inactive in vehicle-treated rats produced a near-maximal activation of LC neurons of rats chronically administered morphine. LC sensitization to CRF was not solely a pharmacological phenomenon but was expressed as hyperresponsivity to physiological stress. Finally, opiate-induced LC sensitization translated to a change in the behavioral repertoire in response to environmental stress (swim stress) such that NE-mediated hyperactive behaviors predominated. The opiate-induced sensitization of the central NE response to stress predicts that chronic opiate administration increases vulnerability to certain stress-related symptoms (e.g., hyperarousal, attentional dysfunction), and this may contribute to the maintenance of opiate-seeking behavior.

Mitochondrial complex I inhibition produces selective damage to hippocampal subfield CA1 in organotypic slice cultures.

The effects of mitochondrial respiratory chain inhibitors and the excitotoxin N-methyl-D-aspartate (NMDA) on cell death in hippocampal subfields CA1 and CA3 were examined in hippocampal organotypic slice cultures. Slice cultures, 2-3 week old, were exposed for 1 h to either the Complex I inhibitors, rotenone or 1-methyl-4-phenylpyridium (MPP+), the Complex II inhibitor 3-nitropropionic acid (3-NP), or the excitotoxin NMDA. Cell death was examined 24 and 48 h following treatment, by measuring propidium iodide (PI) fluorescence. Treatment with 1 micro M Rotenone caused greater cell death in hippocampal subfield CA1 than CA3. Exposure of hippocampal slice cultures to 10 micro M rotenone, to MPP+ or to NMDA resulted in damage to both CA1 and CA3 subfields. 3-NP produced little damage in either subfield. The data suggest that mitochondrial Complex I inhibition can produce selective cell damage in hippocampus and in this regard is similar to that observed following hypoxia/ischemia.

Improvement in neuronal survival after ischemic preconditioning in hippocampal slice cultures.

The main goals of the current study were to assess: (a) whether a sublethal ischemic insult could protect the CA1 subregion of the hippocampus in organotypic slices against a lethal ischemic insult; and (b) whether this protection is long lasting as determined with an accurate immunohistochemical neuronal marker, NeuN. Hippocampal slice cultures were grown for 12-14 days in vitro. Slices were exposed either to oxygen/glucose deprivation (OGD) for 45 min (ischemia), or OGD for 15 min (ischemic preconditioning), 48 h prior to 45 min OGD, or were untreated (sham). Cell death was estimated by propidium iodide fluorescence 1 day after OGD and by NeuN immunohistochemistry 7 days after OGD. Image analysis was employed to measure the relative optical density of the NeuN-signal in all groups. After ischemia, damaged neurons were shrunken or lost and NeuN immunoreactivity was reduced. Relative optical density of NeuN (ROD [NeuN]) was 0.193+/-0.015 in control (sham) (n=9). In slices that underwent ischemia, ROD [NeuN] declined to 0.108+/-0.018 (n=5) in CA1 (*P<0.05 ROD [NeuN] in preconditioned slice cultures was 0.190+/-0.037 (76% higher than the ischemia group). Similar results were found after measuring PI fluorescence. In the CA1 sub-region, PI fluorescence was about 13, 47 and 17% in the sham, ischemic and IPC groups, respectively. We suggest that the immunohistochemical approach validates the dye uptake method used in slice cultures and yields quantitative data specific for neurons. We also conclude that the organotypic hippocampal slice model is useful for studying delayed ischemic preconditioning that is maintained for hours or days after the preconditioning event.