Detection of prions from spiked and free-ranging carnivore feces.

Chronic wasting disease (CWD) is a highly contagious, fatal neurodegenerative disease caused by infectious prions (PrPCWD) affecting wild and captive cervids. Although experimental feeding studies have demonstrated prions in feces of crows (Corvus brachyrhynchos), coyotes (Canis latrans), and cougars (Puma concolor), the role of scavengers and predators in CWD epidemiology remains poorly understood. Here we applied the real-time quaking-induced conversion (RT-QuIC) assay to detect PrPCWD in feces from cervid consumers, to advance surveillance approaches, which could be used to improve disease research and adaptive management of CWD. We assessed recovery and detection of PrPCWD by experimental spiking of PrPCWD into carnivore feces from 9 species sourced from CWD-free populations or captive facilities. We then applied this technique to detect PrPCWD from feces of predators and scavengers in free-ranging populations. Our results demonstrate that spiked PrPCWD is detectable from feces of free-ranging mammalian and avian carnivores using RT-QuIC. Results show that PrPCWD acquired in natural settings is detectable in feces from free-ranging carnivores, and that PrPCWD rates of detection in carnivore feces reflect relative prevalence estimates observed in the corresponding cervid populations. This study adapts an important diagnostic tool for CWD, allowing investigation of the epidemiology of CWD at the community-level.

Leptin activation of dorsal raphe neurons inhibits feeding behavior.

Leptin is a homeostatic regulatory element that signals the presence of energy stores -in the form of adipocytes-which ultimately reduces food intake and increases energy expenditure. Similarly, serotonin (5-HT), a signaling molecule found in both the central and peripheral nervous systems, also regulates food intake. Here we use a combination of pharmacological manipulations, optogenetics, retrograde tracing, and in situ hybridization, combined with behavioral endpoints to physiologically and anatomically identify a novel leptin-mediated pathway between 5-HT neurons in the dorsal raphe nucleus (DRN) and hypothalamic arcuate nucleus (ARC) that controls food intake. In this study, we show that microinjecting leptin directly into the DRN reduces food intake in male Sprague-Dawley rats. This effect is mediated by leptin-receptor expressing neurons in the DRN as selective optogenetic activation of these neurons at either their ARC terminals or DRN cell bodies also reduces food intake. Anatomically, we identified a unique population of serotonergic raphe neurons expressing leptin receptors that send projections to the ARC. Finally, by utilizing in vivo microdialysis and high-performance liquid chromatography, we show that leptin administration to the DRN increases 5-HT efflux into the ARC. Overall, this study identifies a novel circuit for leptin-mediated control of food intake through a DRN-ARC pathway, utilizing 5-HT as a mechanism to control feeding behavior. Characterization of this new pathway creates opportunities for understanding how the brain controls eating behavior, as well as opens alternative routes for the treatment of eating disorders. Significance: Leptin and serotonin both play a vital role in the regulation of food intake, yet there is still uncertainty in how these two molecules interact to control appetite. The purpose of this study is to further understand the anatomical and functional connections between leptin receptor expressing neurons in the dorsal raphe nucleus, the main source of serotonin, and the arcuate nucleus of the hypothalamus, and how serotonin plays a role in this pathway to reduce food intake. Insight gained from this study will contribute to a more thorough understanding of the networks that regulate food intake, and open alternative avenues for the development of treatments for obesity and eating disorders.

Comparative sensitivity of aquatic invertebrate and vertebrate species to wastewater from an operational coal mine in central Queensland, Australia.

Coal excavation and refinement processes generate substantial volumes of contaminated effluent that may be detrimental to aquatic ecosystems. As such, understanding the impacts of coal mine water releases on aquatic animals and ecosystems is essential for effectively managing and protecting neighboring environments. Such information will ultimately be applied towards developing ongoing monitoring strategies that are protective of native wildlife. Despite intensive mining operations in Australia, few studies have documented toxicity associated with coal mine wastewater (CMW) on native species. To address existing knowledge gaps, we investigated acute toxicity (48-96h) using eight native invertebrate species and sub-chronic effects (2 week) using three vertebrate species following exposure to wastewater from two dams (CMW1 and CMW2) located at an open-cut coal mine licensed to discharge into the Fitzroy catchment (Queensland, Australia). Wastewater from these sites is characterized by elevated conductivity, pH, sulfates as well as relatively high total and dissolved metal(loid)s (including As, Al, B, Cu, Mn, Ni, Se and Zn). Acute exposures revealed cladocerans (Daphnia carinata) and planarians (Dugesia sp.) to be the most sensitive species, exhibiting significant mortality after 48 and 96h exposure to CMW2, respectively. Neither wastewater was found to elicit acute toxicity in vertebrates, but a range of sub-lethal morphological effects were observed following the sub-chronic exposures. The overall response pattern was characterized by decreased condition factor and hepatosomatic index in the fish Hypseleotris compressa and Pseudomugil signifier, and in Limnodynastes peronii tadpoles. Tadpoles were generally more sensitive compared to the two fish species. Differences in responses were observed amongst CMW1 and CMW2, which likely relates to differences in physico-chemical properties between sites. Our results have identified several candidate vertebrate and invertebrate species that show promise for ongoing monitoring of water quality and toxicity risk in Central Queensland, Australia.

Effects of coal mine wastewater on locomotor and non-locomotor activities of empire gudgeons (Hypseleotris compressa).

Coal mining represents an important industry in many countries, but concerns exist about the possible adverse effects of minewater releases on aquatic animals and ecosystems. Coal mining generates large volumes of complex wastewater, which often contains high concentrations of dissolved solids, suspended solids, metals, hydrocarbons, salts and other compounds. Traditional toxicological testing has generally involved the assessment of acute toxicity or chronic toxicity with longer-term tests, and while such tests provide useful information, they are poorly suited to ongoing monitoring or rapid assessment following accidental discharge events. As such, there is considerable interest in developing rapid and sensitive approaches to environmental monitoring, and particularly involving the assessment of sub-lethal behavioural responses in locally relevant aquatic species. We therefore investigated behavioural responses of a native Australian fish to coal mine wastewater, to evaluate its potential use for evaluating sub-lethal effects associated with wastewater releases on freshwater ecosystems. Empire gudgeons (Hypseleotris compressa) were exposed to wastewater from two dams located at an open cut coal mine in Central Queensland, Australia and activity levels were monitored using the Multispecies Freshwater Biomonitor® (LimCo International GmbH). A general decrease in locomotor activity (i.e., low frequency movement) and increase in non-locomotor activity (i.e., high frequency movement including ventilation and small fin movement) was observed in exposed fish compared to those in control water. Altered activity levels were observable within the first hour of exposure and persisted throughout the 15-d experiment. Results demonstrate the potential for using behavioural endpoints as tools for monitoring wastewater discharges using native fish species, but more research is necessary to identify responsible compounds and response thresholds, and to understand the relevance of the observed effects for populations in natural receiving environments.

Marine debris is selected as nesting material by the brown booby (Sula leucogaster) within the Swain Reefs, Great Barrier Reef, Australia.

Many seabirds are impacted by marine debris through its presence in foraging and nesting areas. To determine the extent of this problem, marine debris use in nest material of the brown booby (Sula leucogaster) in the Great Barrier Reef, Australia, was investigated. Nine cays were examined using beach and nest surveys. On average, four marine debris items were found per nest (n=96) with 58.3% of surveyed nests containing marine debris. The source of marine debris in nests and transects were primarily oceanic. Hard plastic items dominated both nest (56.8%) and surveyed beaches (72.8%), however only two item types were significantly correlated between these surveys. Nest surveys indicated higher levels of black and green items compared to beach transects. This selectivity for colours and items suggest these nests are not good indicators of environmental loads. This is the first study to examine S. leucogaster nests for marine debris in this location.

Gene therapy for trigeminal pain in mice.

The aim of this study was to test the efficacy of a single direct injection of viral vector encoding for encephalin to induce a widespread expression of the transgene and potential analgesic effect in trigeminal behavioral pain models in mice. After direct injection of herpes simplex virus type 1 based vectors encoding for human preproenkephalin (SHPE) or the lacZ reporter gene (SHZ.1, control virus) into the trigeminal ganglia in mice, we performed an orofacial formalin test and assessed the cumulative nociceptive behavior at different time points after injection of the viral vectors. We observed an analgesic effect on nociceptive behavior that lasted up to 8 weeks after a single injection of SHPE into the trigeminal ganglia. Control virus-injected animals showed nociceptive behavior similar to naive mice. The analgesic effect of SHPE injection was reversed/attenuated by subcutaneous naloxone injections, a µ-opioid receptor antagonist. SHPE-injected mice also showed normalization in withdrawal latencies upon thermal noxious stimulation of inflamed ears after subdermal complete Freund's adjuvant injection, indicating widespread expression of the transgene. Quantitative immunohistochemistry of trigeminal ganglia showed expression of human preproenkephalin after SHPE injection. Direct injection of viral vectors proved to be useful for exploring the distinct pathophysiology of the trigeminal system and could also be an interesting addition to the pain therapists' armamentarium.

Ingestion of marine debris plastic by the wedge-tailed shearwater Ardenna pacifica in the Great Barrier Reef, Australia.

We present the first evidence of ingestion of plastic by seabirds from the southern Great Barrier Reef (GBR), Australia. The occurrence of marine debris ingestion in the wedge-tailed shearwater, Ardenna pacifica, on Heron Island was the focus of this preliminary research. Our findings indicate that 21% of surveyed chicks are fed plastic fragments by their parents, having ingested 3.2 fragments on average. The most common colours of ingested plastic fragments were off/white (37.5%) and green (31.3%). Ingested fragments had a mean size of 10.17±4.55 mm and a mean weight of 0.056±0.051 g. Our results indicate that further research is critical to understanding the extent of ingestion, colour preferences, and what impacts ingestion may have on these and other seabird populations in the GBR.

Noradrenaline increases pain facilitation from the brain during inflammatory pain.

Antidepressants that inhibit the recapture of noradrenaline have variable effects in chronic pain which may be related to the complex role of noradrenaline in pain modulation. Whereas at the spinal cord noradrenaline blocks nociceptive transmission, both antinociception and pronociception were reported after noradrenaline release in the brain. To study the role of noradrenaline in pain modulatory areas of the brain, we elected the dorsal reticular nucleus (DRt), a key pain facilitatory area located at the medulla oblongata. Three studies were performed. First, we show that the infusion in the DRt of nomifensine, which increases local extracellular levels of noradrenaline as shown by in vivo microdialysis, also enhances pain behavioral responses during both phases of the formalin test, a classic inflammatory pain model. Then, we demonstrate that the formalin test triggers the release of noradrenaline in the DRt in a biphasic pattern that matches the two phases of the test. Finally, we show that reducing noradrenaline release into the DRt, using an HSV-1 vector which decreases the expression of tyrosine hydroxylase in noradrenergic DRt-projecting neurons, attenuates pain behavioral responses in both phases of the formalin test. The increased noradrenaline levels induced by the infusion of nomifensine at the DRt, along with the hyperalgesic effects of noradrenaline released at the DRt upon noxious stimulation, indicates that noradrenaline may enhance pain facilitation from the brain. It is important to evaluate if antidepressants that inhibit noradrenaline recapture enhance pain facilitation from the brain herein attenuating their analgesic effects.

Decrease in the expression of N-methyl-D-aspartate receptors in the nucleus tractus solitarii induces antinociception and increases blood pressure.

N-methyl-D-aspartate receptors (NMDAR) have a role in cardiovascular control at the nucleus tractus solitarii (NTS), eliciting increases or decreases in blood pressure (BP), depending on the area injected with the agonists. In spite of the association between cardiovascular control and pain modulation, the effects of manipulating NMDAR in pain responses have never been evaluated. In this study, we decreased the expression of NMDAR in the NTS using gene transfer to target receptor subunits and evaluate long-term effects. Seven days after the injection of lentiviral vectors containing the NR1a subunit cDNA of NMDAR, in antisense orientation, into the intermediate NTS of Wistar rats, BP was measured, and the formalin test of nociception was performed. The antisense vector induced a decrease of NR1 expression in the NTS and elicited BP rises and hypoalgesia. Antisense vectors inhibited formalin-evoked c-Fos expression in the spinal cord, indicating decreased nociceptive activity of spinal neurons. Using a time-course approach, we verified that the onset of both the increases in BP and the hypoalgesia was at 4 days after vector injection into the NTS. The injection of NMDA into the NTS reversed the effects of antisense vectors in pain behavioral responses and spinal neuronal activation and decreased BP and heart rate. The present study shows that the NR1 subunit of the NMDAR at the NTS is critical in the regulation of tonic cardiovascular and nociceptive control and shows an involvement of the nucleus in the modulation of sustained pain.

Reversal of inflammatory pain by HSV-1-mediated overexpression of enkephalin in the caudal ventrolateral medulla.

Targeting supraspinal pain control centers by gene transfer is known to induce sustained analgesia. In this study, we evaluated the effects of injecting a Herpes Simplex Virus type 1 vector which expresses enkephalin (HSV-ENK vector) in the lateralmost part of the caudal ventrolateral medulla (VLMlat), a pain control center that exerts mainly descending inhibitory effects on pain modulation. Overexpression of enkephalin at the VLMlat reduced the number of flinches during the early and delayed phases of the formalin test and decreased c-fos expression in the spinal cord. These antinociceptive effects were detected at 2 and 10days after injection of HSV-ENK in the VLMlat and were completely reversed by local administration of naloxone. Virally driven-enkephalin was expressed from transduced neurons located in the VLMlat and, at lower extent, in the rostral ventromedial medulla. Our results show that HSV-mediated expression of enkephalin in the VLMlat induced antinociceptive effects, likely due to an enhancement of the opioidergic input to the VLMlat which accounted for descending inhibition of the nociceptive transmission at the spinal cord. This study also demonstrates the value of HSV-1 derived vectors to manipulate, in a sustained and directed manner, pain modulatory pathways in the brain, which is important in the study of supraspinal pain control circuits.

Reversal of neuropathic pain by HSV-1-mediated decrease of noradrenaline in a pain facilitatory area of the brain.

Descending modulation of nociceptive transmission depends on the release of noradrenaline at the spinal cord. The role of noradrenaline in the control of nociceptive transmission at the supraspinal pain control system remains understudied. As chronic pain is associated with enhanced descending facilitation of nociceptive transmission, we sought to determine the role of noradrenaline in pain facilitation from the brain during neuropathic pain. We determined the action of the noradrenergic input to the dorsal reticular nucleus (DRt), a unique pain facilitatory area, using the spared nerve injury model. Injections of the α1-adrenoreceptor agonist phenylephrine into the DRt induced hyperalgesia and allodynia, indicating that α1-adrenoreceptors enhance the facilitatory action of the nucleus. This led us to reduce noradrenaline release at the DRt using a viral vector derived from the Herpes Simplex virus type 1 (HSV-1) which carried the tyrosine hydroxylase (TH) transgene in antisense orientation. The reduction of noradrenaline release, confirmed by microdialysis experiments, induced a long-lasting attenuation of pain responses, which was reverted by the local administration of phenylephrine. The present study indicates that the noradrenergic modulation of a pronociceptive area at the supraspinal pain control system accounts for pain facilitation, through the activation of α1-adrenoreceptors. The study also shows that sustained effects on chronic pain can be achieved by decreasing the release of noradrenaline in a pain facilitatory centre of the brain using gene transfer.

Ecological impacts of tributyltin on estuarine communities in the Hastings River, NSW Australia.

Oyster (Saccostrea commercialis) biomonitoring, assessment of oyster and gastropod (Bembicium auratum) abundance, and gastropod imposex were used to measure the significance of tributyltin (TBT) contamination in an intertidal mangrove forest. We studied the bioavailable levels of TBT in oysters approximately 1 km downstream and 2 km upstream from a TBT waste disposal site. We found observable declines in the abundance of oysters and gastropods correlated with the bioavailable TBT and these findings were confirmed by mapping oyster beds. Oyster cover near the disposal site ranged from 0% to 5% while downstream and upstream populations ranged in cover from 25-50% to 5-25%, respectively. Similarly, gastropod abundances at the disposal site were only 7% of the downstream population and 17% of the upstream population. Imposex was present in 90% of female B. auratum from populations near the disposal site but this effect declined more sharply than the population level effects.

Herpes virus-based recombinant herpes vectors: gene therapy for pain and molecular tool for pain science.

This paper reviews work by Yeomans and Wilson in the area of herpes vector-mediated gene transfer to sensory neurons. Beginning in 1997, these researchers have published a number of papers describing and exploiting this technology in altering the phenotype of pain-sensing neurons (nociceptors). Their initial work, continuing to the present, inserted a transgene cassette encoding the human preproenkephalin gene into the thymidine kinase locus under control of a cytomegalovirus promoter. This vector induced enkephalin expression selectively in the nociceptors innervating the tissue onto which it was applied, producing a profound analgesic and antihyperalgesic in acute and chronic pain models in both rodents and non-human primates. An improved version of this vector is now in clinical trials. In addition to inducing the de novo expression of foreign transgenes, this group also investigated the utility of herpes vectors in altering the endogenous genome of nociceptors. Thus, they inserted antisense sequences for genes of interest in the physiology of these neurons and successfully and selectively knocked down expression of several proteins known or thought to be involved in various pain states, including calcitonin gene-related peptide and mu-opioid receptors. They also used similar techniques to investigate the involvement of acid-sensing ion channels and Nav1.7 sodium channel in different pain states. These experiments uniquely allowed for spatially and temporally selective investigations into the function of these proteins in pain, highly valuable information in target validation for therapy development.

PI(3) kinase is associated with a mechanism of immunoresistance in breast and prostate cancer.

Immune escape describes a critical event whereby tumor cells adopt an immunoresistant phenotype to escape adaptive surveillance. We show that expression of a pivotal negative regulator of T-cell function, B7-H1, correlates with PI(3) kinase activation in breast and prostate cancer patients. B7-H1-mediated immunoresistance can be attenuated by inhibitors of the PI(3) kinase pathway, and is dependent on S6K1-mediated translational regulation of B7-H1 protein. Breast and prostate carcinoma cells with activated PI(3) kinase lose the immunoresistant phenotype after treatment with B7-H1 siRNA. Conversely, breast and prostate carcinoma cells with minimal PI(3) kinase activation adopt an immunoresistant phenotype when engineered to overexpress B7-H1 protein. These observations describe a mechanism for immune escape from tumor dormancy in humans that relates to oncogenesis.

Dynamic of migration of HSV-1 from a medullary pronociceptive centre: antinociception by overexpression of the preproenkephalin transgene.

Herpes Simplex Virus type 1 (HSV-1) vectors are known to inhibit nociceptive transmission at the spinal cord after peripheral applications. Similar approaches may also be useful when applied at the supraspinal pain control system as the system includes pronociceptive (facilitatory) components. We performed a study aimed to analyse the migration of HSV-1 along with the inhibition of pronociception from the medullary dorsal reticular nucleus (DRt), a major facilitatory component of the supraspinal pain control system. To study the migration, a HSV-1 vector expressing lacZ under control of the human cytomegalovirus (hCMV) promoter was injected in the DRt and the expression of beta-galactosidase (beta-gal) was detected at 2, 4, 7, 10 and 14 days. Numerous beta-gal-immunoreactive neurons were observed at the injection site until day 4, and at some of the brain areas projecting to the DRt until day 7. To block the pronociceptive effects of the DRt, a HSV-1 vector expressing the preproenkephalin transgene, under the control of hCMV promoter, was injected into the DRt. Behavioural evaluation was performed at the time-points referred above, using the paw withdrawal latency test to evaluate thermal nociceptive responses. Anti-hyperalgesic effects persisted during 4 days, decreasing after that time-point. The present study demonstrates that selective migration of HSV-1 should be considered in gene therapy strategies based on HSV-1 injections into the brain. The study also shows that it is possible to decrease pain facilitation from the brain using opioidergic inhibition of pronociceptive supraspinal areas.

Lentivirus-mediated downregulation of hypothalamic insulin receptor expression.

Regulation of feeding behavior and energy balance are among the central effects of insulin. For example, intracerebroventricular administration of insulin decreases food intake and body weight, whereas antisense oligodeoxynucleotide downregulation of insulin receptors (IRs) produces hyperphagia. To further examine the role of IRs in the central actions of insulin, we designed an IR antisense lentiviral vector (LV-IRAS) and injected this vector into the third ventricle to selectively decrease IR expression in the rat hypothalamus. Three weeks after LV-IRAS administration, the expression of IRs in the hypothalamus was significantly decreased, whereas no changes were observed in hippocampal IR levels. LV-IRAS administration decreased insulin-stimulated phosphorylation of hypothalamic IRs and translocation of the insulin-sensitive glucose transporter GLUT4 in the hypothalamus; no changes in IR signaling were observed in the hippocampus of LV-IRAS-treated rats. Lentivirus-mediated downregulation of IR expression and signaling produced significant increases in body weight, as well as increases in fat mass that were selective for the subcutaneous compartment. Conversely, lean muscle mass and water mass were not affected in LV-IRAS-treated rats compared to rats treated with control virus. Changes in peripheral adiposity were associated with increases in basal hypothalamic leptin signaling in the absence of changes in leptin receptor expression in LV-IRAS rats. Collectively, these data illustrate the important functional relationships between hypothalamic insulin and leptin signaling in the regulation of body composition and provide insight into the mechanisms through which decreases in IR expression and signaling dysregulates leptin activity, thereby promoting increases in peripheral adiposity.

Decrease in inflammatory hyperalgesia by herpes vector-mediated knockdown of Nav1.7 sodium channels in primary afferents.

Induction of peripheral inflammation increases the expression of the Nav1.7 sodium channel in sensory neurons, potentially increasing their excitability. Peripheral inflammation also produces hyperalgesia in humans and an increase in nociceptive responsiveness in animals. To test the relationship between these two phenomena we applied a recombinant herpes simplex-based vector to the hindpaw skin of mice, which encoded both green fluorescent protein (GFP) as well as an antisense sequence to the Nav1.7 gene. The hindpaw was subsequently injected with complete Freund's adjuvant to induce robust inflammation. Application of the vector, but not a control vector encoding only GFP, prevented an increase in Nav1.7 expression in GFP-positive neurons and prevented development of hyperalgesia in both C and Adelta thermonociceptive tests. These results provide clear evidence of the involvement of an increased expression of the Nav1.7 channel in nociceptive neurons in the development of inflammatory hyperalgesia.

Hydrotherapy in labor.

Maternal anxiety and pain prolong labor and contribute to fetal distress. Hydrotherapy during labor may promote relaxation and decrease pain without the risks caused by other treatments. In this pilot study the psychophysiological effects of hydrotherapy on maternal anxiety and pain during labor were examined. Using a randomized, pretest-posttest control group design with repeated measures, 18 term parturients were assigned to a control or an experimental group. Experimental subjects were placed in a tub of 37 degrees C water for 1 hr during early labor. The Wilcoxon two-sample test revealed statistically significant effects. At 15 min bathers' anxiety and pain scores were decreased compared to nonbathers. At 60 min bathers' pain scores were decreased compared to nonbathers. After 15 min of immersion, bathers had a significantly greater increase in plasma volume than nonbathers. No significant differences were found in urine catecholamines or maternal-fetal complications. The small sample limits conclusions, but the findings offer preliminary support for the therapeutic effects of bathing in labor for acute, short-term anxiety and pain reduction.

Genetic therapy for pain management.

Two approaches to genetic therapy for the management of chronic pain have recently been investigated in animal models of pain. First, transgene-mediated delivery of antinociceptive molecules to the cerebrospinal fluid has been performed with engineered cell lines transplanted to the subarachnoid space and with recombinant adenoviruses that transduce pia mater cells. Second, the phenotype of nociceptive neurons has been altered by recombinant herpes viruses overexpressing antinociceptive peptides or reducing expression of endogenous nociceptive molecules. Both approaches attenuate or reverse persistent nociceptive states, suggesting use in the development of genetic therapy for pain management in humans.