Software for designing rigorous and replicable preclinical research: The Experimental Design Accelerator.

In recent years, there have been concerns about research practices in basic and preclinical biomedical research. There have been problems with non-replicable results, and experimental designs lacking internal validity or external or translational validity. The Experimental Design Accelerator (XDA) is Internet-based, interactive software designed to help those trying to design, conduct and document rigorous, replicable and relevant experiments. It leads the investigator step-by-step through a series of decisions that will define the experimental design. It provides background regarding the significance of each decision and the advantages and disadvantages of each possible choice. For example, it leads the researcher to address issues such as choosing a research model, developing testable hypotheses, identifying extraneous variables, dealing with random and systematic error, picking appropriate sample size and picking appropriate statistical analyses. There are also sections to help conduct the experiment consistent with its design and to document the study to facilitate accurate replication. Helpful features include access to an online statistics book and provisions for rapid contact with consulting experts. A number of potential uses for such novel interactive software tools will be discussed.

The impact of supervision on internal medicine residents' attitudes and management of depression in primary care: a pilot study.

OBJECTIVE: The authors examined the effect of supervision on internal medicine residents' attitudes toward and management of depression. METHOD: Internal medicine residents completed a survey during preclinical conferences. The survey included a published, validated questionnaire, the Depression Attitude Questionnaire, and items developed by the researchers. RESULTS: Of residents in attendance on the day of survey administration, 94% (51/54) agreed to participate. The study sample contained 39% of the 139-member residency program. About half (49%) reported feeling uncomfortable managing depression. Perceived training adequacy was correlated with a greater feeling of ease managing depression. Most residents reported screening ≤20% of patients for depression, although 71% indicated they are more likely to screen if it were a priority for their supervisor. Fifty-eight percent indicated that supervisors' attitudes affect their own attitudes. However, significant correlations between supervisor and resident attitudes were not observed. CONCLUSION: The results of this pilot study suggest that supervision can encourage screening and promote resident preparedness to manage depression.

Pimavanserin reverses multiple measures of anxiety in a rodent model of post-traumatic stress disorder.

Pimavanserin is a highly selective 5-HT2A inverse agonist in current medical use. Prior studies suggest that 5-HT2A serotonin receptors may play a role in anxiety and emotional memory. Therefore, pimavanserin was tested in a rat model of PTSD to determine whether it might ameliorate PTSD-like symptoms. The lifetime prevalence of PTSD is estimated to be 125% higher in women than men. Consequently, in an effort to create a robust model of PTSD that was more representative of human PTSD prevalence, 20-week old female rats of the emotionally hyperreactive Lewis strain were used for these studies. The rats were single-housed and exposed twice to restraint stress coupled with predator odor or to a sham-stressed condition. Twenty days after the second stress or sham-stress exposure, rats were injected with saline alone or with 0.3 or 1.0 mg/kg pimavanserin, doses that were confirmed to substantially block 5-HT2A receptor activity in this study without causing any non-specific behavioral or adverse effects. One hour later, rats were tested for anxiety through acoustic startle response, the elevated plus-maze and three parameters of open field behavior. Five days later, blood was sampled for plasma corticosterone. The stressed/saline-injected rats had higher anxiety scores and corticosterone levels than sham-stressed/saline-injected rats. Pimavanserin significantly and generally dose-dependently reversed these persistent stress effects, but had no significant effect on the behavioral measures in normal, non-stressed rats. These results, consistent with a role for the 5-HT2A receptor, suggest that pimavanserin might have potential to reduce some consequences of traumatic stress.

Acute social stress increases biochemical and self report markers of stress without altering spatial learning in humans.

OBJECTIVES: Spatial learning is shown to be influenced by acute stress in both human and other animals. However, the intricacies of this relationship are unclear. Based on prior findings we hypothesized that compared to a control condition, a social stress condition would not affect spatial learning performance despite elevated biochemical markers of stress. METHODS: The present study tested the effects of social stress in human males and females on a subsequent spatial learning task. Social stress induction consisted of evaluative stress (the Trier Social Stress Test, TSST) compared to a placebo social stress. RESULTS: Compared to the placebo condition, the TSST resulted in significantly elevated cortisol and alpha amylase levels at multiple time points following stress induction. In accord, cognitive appraisal measures also showed that participants in the TSST group experienced greater perceived stress compared to the placebo group. However, there were no group differences in performance on a spatial learning task. CONCLUSION: Our findings suggest that unlike physiological stress, social stress does not result in alterations in spatial learning in humans. It is possible that moderate social evaluative stress in humans works to prevent acute stress-mediated alterations in hippocampal learning processes..

The diamidine diminazene aceturate is a substrate for the high-affinity pentamidine transporter: implications for the development of high resistance levels in trypanosomes.

African trypanosomiasis is a disease of humans and livestock in many areas south of the Sahara. Resistance to the few existing drugs is a major impediment to the control of these diseases, and we investigated how resistance to the main veterinary drug diminazene aceturate correlates with changes in drug transport in resistant strains. The strain tbat1(-/-), lacking the TbAT1/P2 aminopurine transporter implicated previously in diminazene transport, was adapted to higher levels of diminazene resistance. The resulting cell line was designated ABR and was highly cross-resistant to other diamidines and moderately resistant to cymelarsan. Procyclic trypanosomes were shown to be a convenient model to study diamidine uptake in Trypanosoma brucei brucei given the lack of TbAT1/P2 and a 10-fold higher activity of the high-affinity pentamidine transporter (HAPT1). Diminazene could be transported by HAPT1 in procyclic trypanosomes. This drug transport activity was lacking in the ABR line, as reported previously for the pentamidine-adapted line B48. The K(m) for diminazene transport in bloodstream tbat1(-/-) trypanosomes was consistent with uptake by HAPT1. Diminazene transport in ABR and B48 cells was reduced compared with tbat1(-/-), but their resistance phenotype was different: B48 displayed higher levels of resistance to pentamidine and the melaminophenyl arsenicals, whereas ABR displayed higher resistance to diminazene. These results establish a loss of HAPT1 function as a contributing factor to diminazene resistance but equally demonstrate for the first time that adaptations other than those determining the initial rates of drug uptake contribute to diamidine and arsenical resistance in African trypanosomes.

Trypanocidal furamidine analogues: influence of pyridine nitrogens on trypanocidal activity, transport kinetics, and resistance patterns.

Current therapies for human African trypanosomiasis (HAT) are unsatisfactory and under threat from emerging drug resistance linked to the loss of transporters, e.g., the P2 aminopurine transporter (TbAT1). Here we compare the uptake and trypanocidal properties of furamidine (DB75), recently evaluated in clinical trials against stage 1 (haemolymphatic) HAT, and two aza analogues, DB820 and CPD0801 (DB829), which are candidate compounds for treatment of stage 2 (neurological) disease. Values of 50% inhibitory concentrations (IC50s) determined in vitro against both wild-type and transporter mutant parasites were submicromolar, with DB75 trypanotoxicity shown to be better than and DB820 trypanotoxicity similar to that of the widely used veterinary trypanocide diminazene, while CPD0801 was less active. Activity correlated with uptake and with the minimum drug exposure time necessary to kill trypanosomes: DB75 accumulated at double and 10-fold the rates of DB820 and CPD0801, respectively. All three compounds inhibited P2-mediated adenosine transport with similar Ki values, indicating affinity values for this permease in the low to submicromolar range. Uptake of DB75, DB820, and CPD0801 was significantly reduced in tbat1-/- parasites and was sensitive to inhibition by adenine, showing that all three compounds are substrates for the P2 transporter. Uptake in vitro was significantly less than that seen with parasites freshly isolated from infected rats, correlating with a downregulation of P2 activity in vitro. We conclude that DB75, DB820, and CPD0801 are actively accumulated by Trypanosoma brucei brucei, with P2 as the main transport route. The aza analogues of DB75 accumulate more slowly than furamidine itself and reveal less trypanocidal activity in standard in vitro drug sensitivity assays.

Academic stress differentially influences perceived stress, salivary cortisol, and immunoglobulin-A in undergraduate students.

Academic examination stress is reported to increase physiological and self-report measures of stress and to decrease immune functioning. Here, we investigate biochemical and self-report measures of stress, immune functioning, and academic pressures before and during a midterm examination period. Undergraduate students were asked to complete a measure of global stress, the perceived stress scale (PSS-10), and to indicate their current level of perceived stress. They also answered questions regarding specific academic pressures and provided a saliva sample for cortisol and salivary immunoglobulin-A (S-IgA) quantification. Students showed increased salivary cortisol concentrations and also reported greater acute perceived stress during the examination period compared to the non-examination period. Although cortisol concentrations and perceived stress were significantly higher during the examination period, participants reported similar levels of global stress (PSS-10) during both testing sessions. Additional analyses showed a non-significant increase in the level of S-IgA from the non-examination period to the examination period. Specific pressure variables that appeared to contribute to stress regulation during the examination week included the amount of time spent studying and concern about the impact of examinations in the future. By demonstrating measures of chronic examination stress, these findings provide new insight into the complex relationship between examination stress, cortisol, and immune functioning.

Experimental sleep fragmentation impairs spatial reference but not working memory in Fischer/Brown Norway rats.

Sleep fragmentation is a common symptom in sleep disorders and other medical complaints resulting in excessive daytime sleepiness. The present study seeks to explore the effects of sleep fragmentation on learning and memory in a spatial reference memory task and a spatial working memory (WM) task. Fischer/Brown Norway rats lived in custom treadmills designed to induce locomotor activity every 2 min throughout a 24-h period. Separate rats were either on a treadmill schedule that allowed for consolidated sleep or experienced no locomotor activation. Rats were tested in one of two water maze-based tests of learning and memory immediately following 24 h of sleep interruption. Rats tested in a spatial reference memory task (eight massed acquisition trials) with a 24-h follow-up probe trial to assess memory retention showed no differences in acquisition performance but were impaired on the 24 h retention of the platform location. In contrast, the performance of rats tested in a spatial WM task (delayed matching to position task) was not impaired. Therefore, sleep fragmentation prior to testing impairs the ability to retain spatial reference memories but does not impair spatial reference memory acquisition or spatial WM in Fischer-Norway rats.

Improving the reliability and availability of railway track switching by analysing historical failure data and introducing functionally redundant subsystems.

Track switches are safety critical assets that not only provide flexibility to rail networks but also present single points of failure. Switch failures within dense-traffic passenger rail systems cause a disproportionate level of delay. Subsystem redundancy is one of a number of approaches, which can be used to ensure an appropriate safety integrity and/or operational reliability level, successfully adopted by, for example, the aeronautical and nuclear industries. This paper models the adoption of a functional redundancy approach to the functional subsystems of traditional railway track switching arrangements in order to evaluate the potential increase in the reliability and availability of switches. The paper makes three main contributions. First, 2P-Weibull failure distributions for each functional subsystem of each common category of points operating equipment are established using a timeline and iterative maximum likelihood estimation approach, based on almost 40,000 sampled failure events over 74,800 years of continuous operation. Second, these results are used as baselines in a reliability block diagram approach to model engineering fault tolerance, through subsystem redundancy, into existing switching systems. Third, the reliability block diagrams are used with a Monte-Carlo simulation approach in order to model the availability of redundantly engineered track switches over expected asset lifetimes. Results show a significant improvement in the reliability and availability of switches; unscheduled downtime reduces by an order of magnitude across all powered switch types, whilst significant increases in the whole-system reliability are demonstrated. Hence, switch designs utilising a functional redundancy approach are well worth further investigation. However, it is also established that as equipment failures are engineered out, switch reliability/availability can be seen to plateau as the dominant contributor to unreliability becomes human error.

A subtype-specific neuropeptide FF receptor antagonist attenuates morphine and nicotine withdrawal syndrome in the rat.

Considerable evidence suggests the Neuropeptide FF (NPFF) and related peptides exert pro-nociceptive and anti-opiate actions, particularly at the supra-spinal level, which may contribute to opiate dependence. The FF1 receptor subtype appears to be primarily responsible for anti-opiate effects. In contrast, stimulation of the FF2 receptor primarily induces pro-opiate effects. AC-262620 is a small molecule, systemically active, selective FF1 receptor antagonist. An initial experiment showed that 10 mg/kg i.p. AC-262620 significantly reduced subsequent naloxone-precipitated somatically expressed withdrawal signs in rats infused s.c. for seven days with 0.3 mg/kg/hr morphine sulfate. A second experiment showed that the same dose of AC-262620 significantly reduced subsequent spontaneous withdrawal signs 23.75 h after termination of seven days s.c. infusion of 0.6 mg/kg/hr morphine sulfate. Chronic nicotine intake may contribute to dependence by overstimulating opiate receptors through release of opiate peptides. By analogy to opiate dependence, it was hypothesized that FF1 receptor activation contributes to nicotine dependence and withdrawal syndrome. AC-262620 significantly reduced somatically expressed withdrawal signs precipitated by the nicotinic antagonist mecamylamine in rats infused for seven days with nicotine bitartrate. Taken together, these findings suggest that NPFF or related neuropeptides contribute to opiate, as well as nicotine, dependence and withdrawal syndrome through the FF1 receptor.

Experimental sleep fragmentation impairs attentional set-shifting in rats.

STUDY OBJECTIVE: To evaluate the effect of experimental sleep fragmentation (sleep interruption; SI) on complex learning in an intradimensional-extradimensional (ID/ED) set-shifting task in rats. DESIGN: A sleep fragmentation paradigm of intermittent forced locomotion was validated in adult rats by examining electrographic effects. Discrimination task performances were assessed in rats following sleep fragmentation or 2 control conditions. PARTICIPANTS: 41 young adult male Fischer-Norway rats. INTERVENTION: A treadmill was used to produce 30 awakenings/h for the 24-h period prior to testing. Exercise control rats received an equivalent amount of treadmill-induced locomotion that permitted 30-minute pauses to allow consolidated sleep. MEASUREMENT AND RESULTS: SI rats were selectively impaired on the extradimensional-shift phase of the task, taking significantly more trials to achieve criterion performance (15.4 +/- 2.0) than either control group (cage control = 10.4 +/- 0.9; exercise control = 6.3 +/- 0.2). The SI schedule reduced the average duration of nonREM sleep (NREMS) episodes to 56 s (baseline = 182 s), while the exercise control group increased average NREMS episode duration to 223 s. Total (24-h) NREMS time declined from 50% during baseline to 33% during SI, whereas rapid eye movement sleep (REMS) was absent in SI animals (7% during baseline and 0% during SI), and time spent awake increased proportionally (from 43% during baseline to 67% during SI). CONCLUSION: 24-hour SI produced impairment in an attentional set-shifting that is comparable to the executive function and cognitive deficits observed in humans with sleep apnea or after a night of experimental sleep fragmentation.

Effects of Sleep Deprivation on Spatial Learning and Memory in Juvenile and Young Adult Rats.

Sleepiness is commonly seen in adolescents and can negatively impact school performance. Little research has investigated the impact of sleepiness in juvenile animals on spatial learning. Sprague-Dawley juvenile (<30 days) and young adult (>60 days) rats were sleep deprived for 24 hours and tested, along with controls, in a water maze task. Sleep deprived juveniles were slower to learn the location of the hidden platform than controls; however, adult performance was not impaired. Sleep deprivation did not impair recall during a probe trial for either age group. Sleep deprivation prior to testing slowed spatial learning in juveniles but not adults.

Validation and scopolamine-reversal of latent learning in the water maze utilizing a revised direct platform placement procedure.

The Morris water maze is routinely used to explore neurobiological mechanisms of working memory. Humans can often acquire working memory relevant to performing a task by mere sensory observation, without having to actually perform the task followed by reinforcement. This can be modeled in the water maze through direct placement of a rat on the escape platform so that it can observe the location, and then assessing the subject's performance in swimming back to the platform. However, direct placement procedures have hardly been studied for two decades, reflecting a controversy about whether direct placement resulted in sufficiently rapid and direct swims back to the platform. In the present study, utilizing revised training methods, a more comprehensive measure of trajectory directness, a more rigorous sham-trained control procedure and an optimal placement-test interval, rats swam almost directly back to the platform in under 4s, significantly more quickly and directly than sham-trained subjects. Muscarinic cholinergic mechanisms, which are inactivated by scopolamine, are essential to memory for standard learning paradigms in the water maze. This experiment determined whether this would also be true for latent learning. ANOVA revealed significant negative effects of scopolamine on both speed and accuracy of trajectory, as well as significant positive effects of direct placement training vs. sham-training. In a probe trial, placement-trained animals without scopolamine spent significantly more time and path length in the target quadrant than trained rats with scopolamine and sham-trained rats without scopolamine. Scopolamine impairments are likely due to effects on memory, since the same dose had little effect on performance with a visible platform. The revised direct placement model offers a means of further comparing the neural mechanisms of latent learning with those of standard instrumental learning.

A re-evaluation of the role of tetrahydropapaveroline in ethanol consumption in rats.

The role of tetrahydropapaveroline (THP), a condensation product of a dopaldehyde with dopamine, in the regulation of alcohol consumption was investigated. In the first experiment, rats received intraventricular injections of either racemic THP hydrobromide (0.65 or 1.3 microg/microl), R-(+)-THP (0.66 or 1.4 microg/microl), or an equal volume of vehicle. The lower doses of both (+/-)-THP and (+)-THP significantly increased volitional alcohol intake. For the racemic compound, the increase was significant at 7-13% concentrations. The R-(+)-enantiomer increased consumption at 4-11 and 15-20% concentrations of ethanol. The higher doses of both compounds did not significantly alter alcohol preference. A second experiment evaluated the chronic effect of THP delivered subcutaneously via osmotic minipump. Animals receiving THP (0.1, 0.5, 1.0, 2.0, and 4.0 mg/ml) did not differ in their alcohol intake, compared to vehicle-treated controls. Whether or not endogenously formed THP participates in the etiology of alcohol addiction remains unclear. Nonetheless, there are few known compounds that induce a preference for unsweetened alcohol solutions over water in laboratory animals.

Modafinil facilitates performance on a delayed nonmatching to position swim task in rats.

Modafinil is a wake-promoting drug approved by the FDA for the treatment of narcolepsy. Recent evidence suggests that modafinil may improve learning and memory processes. To further evaluate possible cognitive properties associated with modafinil, male Sprague-Dawley rats were tested in a delayed nonmatching to position (DNMTP) task. A modified water maze allowed animals to make one of two choices for the location of the escape platform. Each trial consisted of two swims. On the information swim (IS), only one choice was open to the animal for escape. One minute later, a choice swim (CS) presented the animal with two choices with the escape platform in the opposite position. There were 10 trials per day for 10 days. Rats received 0, 30, 55, or 100 mg/kg ip of modafinil 30 min prior to testing. Locomotor activity was also assessed. Animals that received 55 and 100 mg/kg made significantly more correct choices, indicating that higher doses of modafinil learned the task faster than did controls. While animals that received 100 mg/kg did exhibit an enhancement of locomotor activity, this effect did not result in more efficient goal-directed behavior. The evidence is consistent with previous research showing that modafinil facilitates cognitive processes.

Ginkgo biloba extract: cognitive enhancer or antistress buffer.

Constituents extracted from the leaves of the Ginkgo biloba tree possess beneficial properties that may buffer the aging nervous system from deterioration due to oxidative stress. In the present investigation, a standardized extract of G. biloba (EGb 761) or an equal volume of the vehicle was administered (100 mg/kg/day) to senescent (20-month) C57BL/6 male mice for up to 82 consecutive days. Animals were tested twice in the Morris water maze (MWM) after 28 and 70 days of treatment. No differences were observed in acquisition or retention of performance on the water maze. Elevated-plus maze (EPM) trials were conducted prior to and subsequent to the chronic treatment regimen. Marked baseline differences in plus-maze performance were present in the first experiment. A second experiment used a matched-pairs design to minimize preexisting differences. Results supported the hypothesis that EGb 761 may serve as an antistress buffer, attenuating the increase in anxiety typically observed in animals after cold water exposure. Tissue samples from the hippocampus and cortex were analyzed by Western blot for the transcription factor cyclic-AMP response element binding (CREB) protein. EGb 761 had no significant effect on immunoreactivity to CREB from either the hippocampus or the cerebral cortex.

Sleep deprivation impairs recall of social transmission of food preference in rats.

Evidence indicates that sleep plays an important role in learning and memory, and disruption of sleep especially seems to interfere with hippocampal memory processes. Social transmission of food preference (STFP), a natural test of paired associative learning, has been shown to be dependent on the hippocampus. While social transmission of food preference is not a novel task, it has not been used to examine the role of sleep in memory consolidation. Male Sprague-Dawley rats were randomly divided into three groups: cage control; sleep-deprived; and device control. Demonstrator rats were given powdered food mixed with a target spice. Test rats then interacted with demonstrator rats before being given a two choice test of powered food with the target spice or a novel spice. Sleep-deprived rats were then placed in an automated device that prevented sleep for 24 hours. After sleep deprivation, animals were given a preference test again to determine memory for the target spice at both 24 hours and 72 hours. Polysomnography was used to validate the method of sleep deprivation. During immediate preference testing, rats demonstrated a clear preference for the food containing the target spice. Rats that experienced 24 hours of sleep deprivation following the initial testing indicated a significant reduction in the recall of the target spice at 24 and 72 hours. The cage control and device animals maintained their preference for food containing the target spice. Therefore, the loss of sleep interfered with memory consolidation for food preference learned via social transmission.

Risk of obstructive sleep apnea lower in double reed wind musicians.

STUDY OBJECTIVES: Obstructive sleep apnea (OSA) is caused by a collapse of the upper airway. Respiratory muscle training with a wind instrument (didgeridoo) in patients with moderate OSA has been previously shown to improve OSA symptomology. However, a survey of orchestra members did not indicate a difference in OSA risk between wind and non-wind instrumentalist. The present study examines whether playing of different wind instrument types may affect the risk of OSA. METHODS: A national sample of active musicians (n = 906) was surveyed through the internet. Participants' risk for OSA was determined by the Berlin Questionnaire. Additional survey items included questions about general health and musical experience. RESULTS: A binary logistic regression was conducted to determine if OSA risk was predicted by gender, age, number of years playing instrument, number of hours per week playing instrument, and instrument type. Musicians who played a double reed instrument had a lower risk of OSA (p = 0.047) than non-wind instrumentalists. Additionally, in double reed instrumentalists, the number of hours spent playing the instrument predicted lower OSA risk (p = 0.020). The risk for OSA in other wind instruments (i.e., single reed, high brass, and low brass) was not significantly different from non-wind musicians. CONCLUSIONS: Playing a double reed musical instrument was associated with a lower risk of OSA.

Sleep fragmentation reduces hippocampal CA1 pyramidal cell excitability and response to adenosine.

Sleep fragmentation (SF) impairs the restorative/cognitive benefits of sleep via as yet unidentified alterations in neural physiology. Previously, we found that hippocampal synaptic plasticity and spatial learning are impaired in a rat model of SF which utilizes a treadmill to awaken the animals every 2 min, mimicking the frequency of awakenings observed in human sleep apnea patients. Here, we investigated the cellular mechanisms responsible for these effects, using whole-cell patch-clamp recordings. 24h of SF decreased the excitability of hippocampal CA1 pyramidal neurons via decreased input resistance, without alterations in other intrinsic membrane or action potential properties (when compared to cage controls, or to exercise controls that experienced the same total amount of treadmill movement as SF rats). Contrary to our initial prediction, the hyperpolarizing response to bath applied adenosine (30 microM) was reduced in the CA1 neurons of SF treated rats. Our initial prediction was based on the evidence that sleep loss upregulates cortical adenosine A1 receptors; however, the present findings are consistent with a very recent report that hippocampal A1 receptors are not elevated by sleep loss. Thus, increased adenosinergic inhibition is unlikely to be responsible for reduced hippocampal long-term potentiation in SF rats. Instead, the reduced excitability of CA1 pyramidal neurons observed here may contribute to the loss of hippocampal long-term potentiation and hippocampus-dependent cognitive impairments associated with sleep disruption.

A fluorescence-based assay for the uptake of CPD0801 (DB829) by African trypanosomes.

Drug therapies currently used for second stage Human African Trypanosomiasis (HAT) exhibit problems with toxicity, difficulty of administration, and resistance linked to the loss of transporter function. Key to the development of new drugs for HAT is a better understanding of the transport properties of candidate compounds. Standard methods for studying transport utilize radio-labelled permeant or HPLC-MS, however the natural fluorescence of many trypanocidal compounds can be exploited. Here we present a fluorescence-based assay for measuring uptake, by trypanosomes, of CPD0801, a drug candidate for second stage HAT. Sample fluorescence is measured in a 96-well format using a benchtop fluorimeter. Our method is directly applicable to the study of other diamidines with similar fluorescent properties and readily adapted for use with other cell types or fluorescent molecules as we demonstrate for the veterinary trypanocide ethidium.