Pharmacological and behavioral divergence of ketamine enantiomers: implications for abuse liability.

Ketamine, a racemic mixture of (S)-ketamine and (R)-ketamine enantiomers, has been used as an anesthetic, analgesic and more recently, as an antidepressant. However, ketamine has known abuse liability (the tendency of a drug to be used in non-medical situations due to its psychoactive effects), which raises concerns for its therapeutic use. (S)-ketamine was recently approved by the United States' FDA for treatment-resistant depression. Recent studies showed that (R)-ketamine has greater efficacy than (S)-ketamine in preclinical models of depression, but its clinical antidepressant efficacy has not been established. The behavioral effects of racemic ketamine have been studied extensively in preclinical models predictive of abuse liability in humans (self-administration and conditioned place preference [CPP]). In contrast, the behavioral effects of each enantiomer in these models are unknown. We show here that in the intravenous drug self-administration model, the gold standard procedure to assess potential abuse liability of drugs in humans, rats self-administered (S)-ketamine but not (R)-ketamine. Subanesthetic, antidepressant-like doses of (S)-ketamine, but not of (R)-ketamine, induced locomotor activity (in an opioid receptor-dependent manner), induced psychomotor sensitization, induced CPP in mice, and selectively increased metabolic activity and dopamine tone in medial prefrontal cortex (mPFC) of rats. Pharmacological screening across thousands of human proteins and at biological targets known to interact with ketamine yielded divergent binding and functional enantiomer profiles, including selective mu and kappa opioid receptor activation by (S)-ketamine in mPFC. Our results demonstrate divergence in the pharmacological, functional, and behavioral effects of ketamine enantiomers, and suggest that racemic ketamine's abuse liability in humans is primarily due to the pharmacological effects of its (S)-enantiomer.

Evidence in primates supporting the use of chemogenetics for the treatment of human refractory neuropsychiatric disorders.

Non-human primate (NHP) models are essential for developing and translating new treatments that target neural circuit dysfunction underlying human psychopathology. As a proof-of-concept for treating neuropsychiatric disorders, we used a NHP model of pathological anxiety to investigate the feasibility of decreasing anxiety by chemogenetically (DREADDs [designer receptors exclusively activated by designer drugs]) reducing amygdala neuronal activity. Intraoperative MRI surgery was used to infect dorsal amygdala neurons with AAV5-hSyn-HA-hM4Di in young rhesus monkeys. In vivo microPET studies with [11C]-deschloroclozapine and postmortem autoradiography with [3H]-clozapine demonstrated selective hM4Di binding in the amygdala, and neuronal expression of hM4Di was confirmed with immunohistochemistry. Additionally, because of its high affinity for DREADDs, and its approved use in humans, we developed an individualized, low-dose clozapine administration strategy to induce DREADD-mediated amygdala inhibition. Compared to controls, clozapine selectively decreased anxiety-related freezing behavior in the human intruder paradigm in hM4Di-expressing monkeys, while coo vocalizations and locomotion were unaffected. These results are an important step in establishing chemogenetic strategies for patients with refractory neuropsychiatric disorders in which amygdala alterations are central to disease pathophysiology.

Manure ammonia and hydrogen sulfide emissions from a Western dairy storage basin.

The reporting of ammonia (NH) and hydrogen sulfide (HS) emissions from dairies to the federal government depends on the magnitude of the emissions. However, little is known about their daily NH and HS emissions and what influences those emissions. Emissions of NH and HS from two manure storage basins at a 4400-head western free-stall dairy were measured intermittently over 2 yr. Each basin went through stages of filling, drying, and then removal of the manure during the study period. Emissions were determined using backward Lagrangian Stochastic and vertical radial plume methods. Ammonia emissions ranged from 35 to 59 kg d in one basin and from 86 to 90 kg d in a second basin, corresponding to a range of 7 to 19 g d head. Basin NH emissions were highest during initial filling and when the manure was removed. Mean HS emissions ranged from 5 to 22 kg d (1.1-4.6 g d head). Basin HS emissions were highest when the basin was filling. Crusting of the basin surface reduced NH but not HS emissions. The cessation of basin filling reduced HS but not NH emissions. Air temperature and wind conditions were correlated with NH emissions. Barometric pressure decreases were correlated with episodic HS emissions. The variability in emissions with stage of manure handling and storage and meteorological conditions indicates that determining the maximum daily emissions and the annual emissions from such waste basins requires consideration of each stage in conjunction with the climatic conditions during the stage.

Inhomogeneity of methane emissions from a dairy waste lagoon.

UNLABELLED: Methane (CH4) is the dominant greenhouse gas emitted by animal agriculture manure. Since the gas is relatively insoluble in water, it is concentrated in discrete bubbles that rise through waste lagoons and burst at the surface. This results in lagoon emissions that are inhomogeneous in both space and time. Emissions from a midwestern dairy waste lagoon were measured over 2 weeks to evaluate the spatial homogeneity of the source emissions and to compare two methods for measuring this inhomogeneous emission. Emissions were determined using an inverse dispersion model based on CH4 concentrations measured both by a single scanning tunable diode laser (TDL) aimed at a series of reflectors and by flame ionization detection (FID) gas chromatography on line-sampled air. Emissions were best estimated using scanned TDL concentrations over relatively short optical paths that collectively span the entire cross-wind width of the source, so as to provide both the best capture of discrete plumes from the bursting bubbles on the lagoon surface and the best detection of CH4 background concentrations. The lagoon emissions during the study were spatially inhomogeneous at hourly time scales. Partitioning the inhomogeneous source into two source regions reduced the estimated emissions of the overall lagoon by 57% but increased the variability. Consequently, it is important to assess the homogeneity of a source prior to measurements and final emissions calculation. IMPLICATIONS: Plans for measuring methane emissions from waste lagoons must take into account the spatial inhomogeneity of the source strength. The assumption of emission source homogeneity for a low-solubility gas such as CH4 emitted from an animal waste lagoon can result in significant emission overestimates. The entire breadth and length of the area source must be measured, preferably with multiple optical paths, for the detection of discrete plumes from the different emitting regions and for determining the background concentration. Other gases with similarly poor solubility in water may also require partitioning of the lagoon source area.

Hydrogen Sulfide Emissions from Sow Farm Lagoons across Climates Zones.

Hydrogen sulfide (HS) emissions were measured periodically over the course of 2 yr at three sow waste lagoons representing humid mesothermal (North Carolina, NC), humid microthermal (Indiana, IN), and semiarid (Oklahoma, OK) climates. Emissions were determined using a backward Lagrangian stochastic model in conjunction with line-sampled HS concentrations and measured turbulence. The median annual sow-specific (area-specific) lagoon emissions at the OK farm were approximately 1.6 g head [hd] d (5880 µg m s), whereas those at the IN and NC sow farms were 0.035 g hd d (130 µg m s), and 0.041 g hd d (260 µg m s), respectively. Hydrogen sulfide emissions generally increased with wind speed. The daily HS emissions from the OK lagoon were greatest during the first half of the year and decreased as the year progressed. Emissions were episodic at the NC and IN lagoons. The generally low emissions at the NC and IN lagoons were probably a result of significant populations of purple sulfur bacteria maintained in the humid mesothermal and humid microthermal climates. Most of the large HS emission events at the NC and IN lagoons appeared to be a result of either precipitation events or liquid pump-out events. The high emissions at the OK lagoon in a semiarid climate were largely a result of high wind speeds enhancing both lagoon and air boundary layer mixing. The climate (air temperature, winds, and precipitation) appeared to influence the HS emissions from lagoons.

Effects of atmospheric and manure surface conditions on H2 S emissions from an in-ground finisher hog manure slurry tank.

Hydrogen sulfide (H2 S) emitted by livestock operations can be detrimental to human health. The storage of hog manure is a significant agricultural source of H2 S emissions. H2 S emissions from a ground-level Midwestern hog finisher manure tank were measured for 8-20 days each quarter over a 15-month period. After excluding 4 days with outlier emissions the mean daily emission was 1.89 g H2 S m-2 day-1 . Mean daily emission was 1.39 g H2 S m-2 day-1 when the slurry surface was liquid and 3.00 g H2 S m-2 day-1 when crusted. Emissions however were not significantly different whether the surface was liquid or crusted when differences in temperature were considered. Diurnal variation in emissions was not correlated with air temperature, water vapor saturation deficit, or wind speed when the manure surface was crusted but was positively correlated with these variables when the surface was not crusted. Daily H2 S emissions were modeled according to two-film theory incorporating resistance approach with limited success. Additional emissions measurements with greater documentation of the manure liquid composition and crust characteristics are needed to assess the component transport resistances in the emissions model.

Expression of the excitatory opsin ChRERα can be traced longitudinally in rat and nonhuman primate brains with PET imaging.

Optogenetics is a widely used technology with potential for translational research. A critical component of such applications is the ability to track the location of the transduced opsin in vivo. To address this problem, we engineered an excitatory opsin, ChRERα (hChR2(134R)-V5-ERα-LBD), that could be visualized using positron emission tomography (PET) imaging in a noninvasive, longitudinal, and quantitative manner. ChRERα consists of the prototypical excitatory opsin channelrhodopsin-2 (ChR2) and the ligand-binding domain (LBD) of the human estrogen receptor α (ERα). ChRERα showed conserved ChR2 functionality and high affinity for [18F]16α-fluoroestradiol (FES), an FDA-approved PET radiopharmaceutical. Experiments in rats demonstrated that adeno-associated virus (AAV)-mediated expression of ChRERα enables neural circuit manipulation in vivo and that ChRERα expression could be monitored using FES-PET imaging. In vivo experiments in nonhuman primates (NHPs) confirmed that ChRERα expression could be monitored at the site of AAV injection in the primary motor cortex and in long-range neuronal terminals for up to 80 weeks. The anatomical connectivity map of the primary motor cortex identified by FES-PET imaging of ChRERα expression overlapped with a functional connectivity map identified using resting state fMRI in a separate cohort of NHPs. Overall, our results demonstrate that ChRERα expression can be mapped longitudinally in the mammalian brain using FES-PET imaging and can be used for neural circuit modulation in vivo.

DREADD-mediated amygdala activation is sufficient to induce anxiety-like responses in young nonhuman primates.

Anxiety disorders are among the most prevalent psychiatric disorders, with symptoms often beginning early in life. To model the pathophysiology of human pathological anxiety, we utilized Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) in a nonhuman primate model of anxious temperament to selectively increase neuronal activity of the amygdala. Subjects included 10 young rhesus macaques; 5 received bilateral infusions of AAV5-hSyn-HA-hM3Dq into the dorsal amygdala, and 5 served as controls. Subjects underwent behavioral testing in the human intruder paradigm following clozapine or vehicle administration, prior to and following surgery. Behavioral results indicated that clozapine treatment post-surgery increased freezing across different threat-related contexts in hM3Dq subjects. This effect was again observed approximately 1.9 years following surgery, indicating the long-term functional capacity of DREADD-induced neuronal activation. [11C]deschloroclozapine PET imaging demonstrated amygdala hM3Dq-HA specific binding, and immunohistochemistry revealed that hM3Dq-HA expression was most prominent in basolateral nuclei. Electron microscopy confirmed expression was predominantly on neuronal membranes. Together, these data demonstrate that activation of primate amygdala neurons is sufficient to induce increased anxiety-related behaviors, which could serve as a model to investigate pathological anxiety in humans.

DREADD-mediated amygdala activation is sufficient to induce anxiety-like responses in young nonhuman primates.

Anxiety disorders are among the most prevalent psychiatric disorders, with symptoms often beginning early in life. To model the pathophysiology of human pathological anxiety, we utilized Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) in a nonhuman primate model of anxious temperament to selectively increase neuronal activity of the amygdala. Subjects included 10 young rhesus macaques; 5 received bilateral infusions of AAV5-hSyn-HA-hM3Dq into the dorsal amygdala, and 5 served as controls. Subjects underwent behavioral testing in the human intruder paradigm following clozapine or vehicle administration, prior to and following surgery. Behavioral results indicated that clozapine treatment post-surgery increased freezing across different threat-related contexts in hM3Dq subjects. This effect was again observed approximately 1.9 years following surgery, indicating the long-term functional capacity of DREADD-induced neuronal activation. [11C]deschloroclozapine PET imaging demonstrated amygdala hM3Dq-HA specific binding, and immunohistochemistry revealed that hM3Dq-HA expression was most prominent in basolateral nuclei. Electron microscopy confirmed expression was predominantly on neuronal membranes. Together, these data demonstrate that activation of primate amygdala neurons is sufficient to induce increased anxiety-related behaviors, which could serve as a model to investigate pathological anxiety in humans.

Emissions of hydrogen sulfide from a western open-lot dairy.

Hydrogen sulfide (H2 S) emissions are considered hazardous to the environment. Animal agriculture operations must therefore report emissions exceeding a threshold to the National Response Center. Estimating the emissions from operations is needed because monitoring at all operations is not possible. However, little is known about H2 S emissions from open-lot dairy operations commonly found in dry regions. Daily mean H2 S emissions from a Texas open-lot dairy were relatively uniform over the year. Emissions were greatest during the fall when air temperatures were relatively high and vapor pressure deficits were low. Higher air temperatures and wind speeds and lower surface wetness corresponded with higher mean hourly emissions in winter, spring, and summer. Hourly mean emissions in the fall differed substantially from those of the other seasons. These high fall emissions appear to have been associated with unreported producer activity of hauling manure from the manure windrow storage onto the surrounding fields. Mean annual live animal basis emissions were 3.6 g d-1 hd-1 . Annual mean emissions for the entire 3,400 head dairy were 12 kg d-1 . The maximum measured daily emissions for this 3,492-cow dairy were 33.1 kg d-1 .

Hydrogen sulfide emissions from a midwestern manure slurry storage basin.

Hydrogen sulfide (H2 S) emissions from midwestern U.S. dairy lagoons are episodic and seasonal. Emissions were determined using an inverse diffusion model in conjunction with measured upwind and downwind line-averaged H2 S concentrations and turbulence. Mean daily H2 S emissions from manure stored in earthen basins was 0.97 µg m-2 s-1 (σ = 1.35 µg m-2 s-1 ). Mean live animal basis daily emission from the basins was 1.1 g d-1 hd-1 (0.84 g d-1 AU-1 ). Daily emission was modeled using the van't Hoff function with air temperature as a surrogate for slurry surface temperature and a linear function of friction velocity. The mean standard error of estimate of the model was 1.8 µg m-2 s-1 (2.0 g d-1 hd-1 , 1.6 g d-1 AU-1 ) and accounted for 60% of emissions variability. H2 S emissions were enhanced for short periods during the year when the stored slurry was loaded onto trucks for removal. Emissions from the basins were not statistically different as barn manure handling changed from flushing to scraping. More measurements are needed to verify annual emissions estimates for these manure slurry storage basins and derive emission factors for these slurry storage systems.

Variation in hydrogen sulfide emissions from a U.S. Midwest anaerobic dairy lagoon.

Hydrogen sulfide (H2 S) emissions from midwestern United States dairy anaerobic waste lagoons are episodic and seasonal. Emissions were modeled using an inverse diffusion model in conjunction with measured concentrations and turbulence. The potential for lagoon mixing was estimated by the Brunt frequency using a theoretical Fourier series temperature profile model constructed from measured air and lagoon temperatures. Annual H2 S emissions from the dairy parlor and holding area liquid waste, based on 318 d of measurement, were 212 g m-2 or 807 g head of cattle-1 . Hydrogen sulfide emissions were highest in the spring and the fall. Eleven days with emissions >7 g d-1 head-1 accounted for 25% of the annual emissions. Shear mixing appeared to dominate the mixing in the lagoon when the lagoon was estimated to be nearly isothermal. Wind shear correlated with significantly greater daily mean emissions. The H2 S emissions from this lagoon appeared to result from a series of processes; biogenic production of H2 S in the sludge, H2 S-enriched bubbles rise through the lagoon by buoyancy and wind shear induced mixing, and bubbles bursting at the surface either due to desiccation of the bubbles or surface disturbances induced by wind and precipitation.

Translational PET applications for brain circuit mapping with transgenic neuromodulation tools.

Transgenic neuromodulation tools have transformed the field of neuroscience over the past two decades by enabling targeted manipulation of neuronal populations and circuits with unprecedented specificity. Chemogenetic and optogenetic neuromodulation systems are among the most widely used and allow targeted control of neuronal activity through the administration of a selective compound or light, respectively. Innovative genetic targeting strategies are utilized to transduce specific cells to express transgenic receptors and opsins capable of manipulating neuronal activity. These allow mapping of neuroanatomical projection sites and link cellular manipulations with brain circuit functions and behavior. As these tools continue to expand knowledge of the nervous system in preclinical models, developing translational applications for human therapies is becoming increasingly possible. However, new strategies for implementing and monitoring transgenic tools are needed for safe and effective use in translational research and potential clinical applications. A major challenge for such applications is the need to track the location and function of chemogenetic receptors and opsins in vivo, and new developments in positron emission tomography (PET) imaging techniques offer promising solutions. The goal of this review is to summarize current research combining transgenic tools with PET for in vivo mapping and manipulation of brain circuits and to propose future directions for translational applications.

18F-labeled radiotracers for in vivo imaging of DREADD with positron emission tomography.

Designer Receptors Exclusively Activated by Designer Drugs (DREADD) are a preclinical chemogenetic approach with clinical potential for various disorders. In vivo visualization of DREADDs has been achieved with positron emission tomography (PET) using 11C radiotracers. The objective of this study was to develop DREADD radiotracers labeled with 18F for a longer isotope half-life. A series of non-radioactive fluorinated analogs of clozapine with a wide range of in vitro binding affinities for the hM3Dq and hM4Di DREADD receptors has been synthesized for PET. Compound [18F]7b was radiolabeled via a modified 18F-deoxyfluorination protocol with a commercial ruthenium reagent. [18F]7b demonstrated encouraging PET imaging properties in a DREADD hM3Dq transgenic mouse model, whereas the radiotracer uptake in the wild type mouse brain was low. [18F]7b is a promising long-lived alternative to the DREADD radiotracers [11C]clozapine ([11C]CLZ) and [11C]deschloroclozapine ([11C]DCZ).

High-potency ligands for DREADD imaging and activation in rodents and monkeys.

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a popular chemogenetic technology for manipulation of neuronal activity in uninstrumented awake animals with potential for human applications as well. The prototypical DREADD agonist clozapine N-oxide (CNO) lacks brain entry and converts to clozapine, making it difficult to apply in basic and translational applications. Here we report the development of two novel DREADD agonists, JHU37152 and JHU37160, and the first dedicated 18F positron emission tomography (PET) DREADD radiotracer, [18F]JHU37107. We show that JHU37152 and JHU37160 exhibit high in vivo DREADD potency. [18F]JHU37107 combined with PET allows for DREADD detection in locally-targeted neurons, and at their long-range projections, enabling noninvasive and longitudinal neuronal projection mapping.

Specific dynamic action of ambystomatid salamanders and the effects of meal size, meal type, and body temperature.

The past decade has witnessed a dramatic increase in studies of amphibian and reptile specific dynamic action (SDA). These studies have demonstrated that SDA, the summed energy expended on meal digestion and assimilation, is affected significantly by meal size, meal type, and body size and to some extent by body temperature. While much of this attention has been directed at anuran and reptile SDA, we investigated the effects of meal size, meal type, and body temperature on the postprandial metabolic responses and the SDA of the tiger salamander (Ambystoma tigrinum tigrinum). We also compared the SDA responses among six species of Ambystoma salamanders representing the breadth of Ambystoma phylogeny. Postprandial peaks in VO(2) and VO(2), duration of elevated metabolism, and SDA of tiger salamanders increased with the size of cricket meals (2.5%-12.5% of body mass). For A. tigrinum, as for other ectotherms, a doubling of meal size results in an approximate doubling of SDA, a function of equal increases in peak VO(2) and duration. For nine meal types of equivalent size (5% of body mass), the digestion of hard-bodied prey (crickets, superworms, mealworms, beetles) generated larger SDA responses than the digestion of soft-bodied prey (redworms, beetle larvae). Body temperature affected the profile of postprandial metabolism, increasing the peak and shortening the duration of the profile as body temperature increased. SDA was equivalent among three body temperatures (20 degrees, 25 degrees, and 30 degrees C) but decreased significantly at 15 degrees C. Comparatively, the postprandial metabolic responses and SDA of Ambystoma jeffersonianum, Ambystoma maculatum, Ambystoma opacum, Ambystoma talpoideum, Ambystoma texanum, and the conspecific Ambystoma tigrinum mavortium digesting cricket meals that were 5% of their body mass were similar (independent of body mass) to those of A. t. tigrinum. Among the six species, standard metabolic rate, peak postprandial VO(2), and SDA scaled with body mass with mass exponents of 0.72, 0.78, and 1.05, respectively.

Depression, anxiety, and tobacco use: Overlapping impediments to sleep in a national sample of college students.

OBJECTIVES: To examine how tobacco use and depression/anxiety disorders are related to disturbed sleep in college students. PARTICIPANTS: 85,138 undergraduate respondents (66.3% female, 74.5% white, non-Hispanic, ages 18-25) from the Spring 2011 American College Health Association-National College Health Assessment II database. METHODS: Multivariate analyses of tobacco use (none, intermediate, daily) and mental health (diagnosed and/or symptomatic depression or anxiety) were used to predict sleep disturbance. RESULTS: Daily tobacco use was associated with more sleep problems than binge drinking, illegal drug use, obesity, gender, and working >20 hours/week. Students with depression or anxiety reported more sleep disturbances than individuals without either disorder, and tobacco use in this population was associated with the most sleep problems. CONCLUSIONS: Tobacco use and depression/anxiety disorders are both independently associated with more sleep problems in college students. Students with depression and/or anxiety are more likely to be daily tobacco users, which likely exacerbates their sleep problems.

Proper laboratory notebook practices: protecting your intellectual property.

A laboratory notebook contains a wealth of knowledge that can be critical for establishing evidence in support of intellectual property rights and for refuting claims of research misconduct. The proper type, organization, use, maintenance, and storage of laboratory notebooks should be a priority for everyone at research institutions. Failure to properly document research activities can lead to serious problems, including the loss of valuable patent rights. Consequences of improper laboratory notebook practices can be harsh; numerous examples are described in court cases and journal articles, indicating a need for research institutions to develop strict policies on the proper use and storage of research documentation.