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.

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.

Passive immunization against nicotine attenuates somatic nicotine withdrawal syndrome in the rat.

INTRODUCTION: Nicotine immunization is under consideration as an intervention for smoking cessation. Therefore, it was of interest to evaluate the effects of nicotine antibodies on the withdrawal syndrome following termination of chronic nicotine administration. METHODS: Experiment 1 determined whether passive immunization following continuous nicotine infusion would alter the intensity of nicotine withdrawal syndrome in the rat. Fourteen rats were rendered nicotine dependent by 7 days of subcutaneous nicotine bitartrate infusion. On the final day, seven rats received 150 mg intraperitoneal (i.p.) of immune gamma globulin (IgG) raised against 3'-aminomethylnicotine-recombinant Pseudomonas aeruginosa exoprotein A (NicVAX, Nabi Biopharmaceuticals, Rockville, MD) and seven rats received normal IgG. Rats were observed under blind conditions for somatically expressed nicotine abstinence signs immediately prior to drug termination and at 12, 24, and 36 hr afterward. In Experiment 2, similarly treated rats were observed at 6- and 72-hr postwithdrawal, to test the possibility that immunization altered the time course rather than the intensity of withdrawal syndrome. Experiment 3 tested whether immunized rats were still nicotine dependent. Without pump removal, each rat was challenged by 1/mg/kg mecamylamine HCl and observed for precipitated withdrawal syndrome. RESULTS: In Experiment 1, there was no premature withdrawal syndrome during nicotine infusion. After termination, the immunized group had significantly fewer withdrawal signs than controls. Experiment 2 showed that immunization did not simply alter the timing of the nicotine abstinence syndrome since immunization did not increase signs before or after the usual withdrawal timeframe. In Experiment 3, rats immunized on the final day of infusion were still nicotine dependent since they exhibited a vigorous mecamylamine-precipitated withdrawal syndrome. DISCUSSION: Nicotine antibodies did not precipitate a withdrawal syndrome, and they markedly reduced the severity of spontaneous nicotine withdrawal. The present data suggests that this may be most readily explained by their reported delay of nicotine clearance.

Rodent models of nicotine withdrawal syndrome.

Simple, rapid and inexpensive rodent models of nicotine physical dependence and withdrawal syndrome have proved useful for preliminary screening of smoking cessation treatments. They have led to an exponential increase of knowledge regarding the underlying neurobiological mechanisms of dependence and withdrawal syndrome. The human nicotine withdrawal syndrome in smoking cessation is variable and multidimensional, involving irritability, anxiety, depression, cognitive and attentional impairments, weight gain, sleep disturbances, and craving for nicotine. Aside from sleep disturbances, analogous phenomena have been seen in rodent models using different measures of withdrawal intensity. It appears likely that different withdrawal phenomena may involve some partially divergent mechanisms. For example, depression-like phenomena may involve alterations in mechanisms such as the mesolimbic dopamine pathway from the ventral tegmental area to the nucleus accumbens. Irritability and anxiety may involve alterations in endogenous opioid systems and other regions, such as the amygdala. This chapter reviews many additional anatomical, neurochemical, and developmental elements that impact nicotine physical dependence.

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.

Bupropion attenuates nicotine abstinence syndrome in the rat.

RATIONALE: Bupropion reduces discomfort and craving associated with smoking cessation. This study determined whether a rat model of nicotine dependence could detect such nicotine abstinence-alleviating effects. OBJECTIVES: Experiments determined whether the abstinence-alleviating effects of bupropion were detectable by (1) behavioral abstinence signs precipitated by the nicotinic antagonist mecamylamine, (2) place aversion conditioned to mecamylamine-precipitated nicotine abstinence, and (3) spontaneous behavioral abstinence signs after abrupt nicotine withdrawal. METHODS: In experiments 1 and 2, nicotine-dependent rats were coinfused for 7 days with 3.15 mg/kg/day nicotine and 20 mg/kg/day bupropion or with nicotine alone. They were then challenged with 1 mg/kg mecamylamine and observed for behavioral abstinence signs (experiment 1) or place aversion conditioned to precipitated abstinence (experiment 2). In experiment 3, rats were nicotine-infused for 7 days as above. A day after termination of nicotine infusion, rats were observed for spontaneous nicotine abstinence signs before and after injection with saline or bupropion. RESULTS: In experiment 1, rats coinfused with nicotine and bupropion had significantly fewer mecamylamine-precipitated abstinence signs than rats infused with nicotine alone but similar numbers to rats infused with saline alone. In experiment 2, bupropion pretreatment significantly reduced the aversiveness of mecamylamine-precipitated nicotine abstinence. In experiment 3, a single bupropion injection dose-dependently alleviated spontaneous nicotine abstinence syndrome. CONCLUSIONS: These results suggest that these rat models of nicotine dependence and abstinence syndrome may be useful in detecting nicotine abstinence-alleviating effects of potential medications for smoking cessation. The effects of acute bupropion administration raise interesting questions regarding bupropion's mechanism of action.

Reversal of morphine tolerance by a compound with NPFF receptor subtype-selective actions.

Neuropeptide FF (NPFF) modulates opiate actions. It has pro-nociceptive effects, primarily through the NPFF receptor 1 subtype, and anti-nociceptive effects, primarily through the NPFFR2 subtype. AC-263093 is a small l, organic, systemically active molecule that was previously shown to functionally activate NPFFR2, but not NPFFR1. It was hypothesized that AC-263093 would attenuate morphine tolerance. Rats were tested for radiant heat tail-flick latency before and after 5 mg/kg morphine sulfate s.c. They were then rendered morphine-tolerant by continuous subcutaneous infusion of 17.52 mg/kg/day morphine sulfate. On the seventh day of infusion, they were retested for analgesia 10 and 20 min after 5mg/kg morphine sulfate s.c. Tolerance was indicated by reduction of morphine analgesia from the pre-infusion test. Fifty minutes prior to morphine challenge, rats received either 10 mg/kg i.p. AC-263093 or injection vehicle alone. AC-2623093-treated rats had far smaller tolerance scores than control rats. This drug effect was significant, p = 0.015. The same dose of AC-263093 had almost no analgesic effect in non-tolerant, saline-infused rats. In vitro experiments revealed that AC-263093 had equal affinity for NPFFR1 and NPFFR2, and functionally inactivated NPFFR1, in addition to its previously shown ability to activate NPFFR2. Thus, altering the balance between activation of NPFF receptor subtypes may provide one approach to reversing opiate tolerance.

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.

Passive immunization against nicotine attenuates nicotine discrimination.

Ten rats were trained in a two lever operant chamber to press different levers after a nicotine injection (0.14 mg/kg s.c.) or a saline injection on an FR10 schedule. The rats were then injected i.p. with either 150 mg nicotine-specific IgG or the same amount of control IgG from non-immunized rabbits. On successive days, they were retested with both levers active after a saline injection, a full training dose of nicotine and a half dose of nicotine (0.07 mg/kg s.c.). After saline injection, both groups pressed the saline lever almost exclusively. After each of the nicotine doses, the immunized rats performed a significantly lower percentage of their lever presses on the nicotine lever than did non-immunized rats. The results suggest that passive immunization can interfere with the stimulus properties of nicotine.

Short-term blueberry-enriched diet prevents and reverses object recognition memory loss in aging rats.

OBJECTIVE: Previously, 4 mo of a blueberry-enriched (BB) antioxidant diet prevented impaired object recognition memory in aging rats. Experiment 1 determined whether 1- and 2-mo BB diets would have a similar effect and whether the benefits would disappear promptly after terminating the diets. Experiment 2 determined whether a 1-mo BB diet could subsequently reverse existing object memory impairment in aging rats. METHODS: In experiment 1, Fischer-344 rats were maintained on an appropriate control diet or on 1 or 2 mo of the BB diet before testing object memory at 19 mo postnatally. In experiment 2, rats were tested for object recognition memory at 19 mo and again at 20 mo after 1 mo of maintenance on a 2% BB or control diet. RESULTS: In experiment 1, the control group performed no better than chance, whereas the 1- and 2-mo BB diet groups performed similarly and significantly better than controls. The 2-mo BB-diet group, but not the 1-mo group, maintained its performance over a subsequent month on a standard laboratory diet. In experiment 2, the 19-mo-old rats performed near chance. At 20 mo of age, the rats subsequently maintained on the BB diet significantly increased their object memory scores, whereas the control diet group exhibited a non-significant decline. The change in object memory scores differed significantly between the two diet groups. CONCLUSION: These results suggest that a considerable degree of age-related object memory decline can be prevented and reversed by brief maintenance on BB diets.

Effect of a total smoking ban in a maximum security psychiatric hospital.

An archival study was performed in a maximum security forensic hospital to evaluate the effects of a total ban on smoking and all tobacco products. One hundred and forty patients were characterized as nonsmokers or light, moderate or heavy smokers. Patient records for the four weeks prior to the ban were compared with their records for the four weeks subsequent to the ban. Numbers of sick calls, total disruptive behaviors and verbal aggression declined markedly and significantly following the ban in those patients previously classified as moderate or heavy smokers. Weight increased significantly, but almost equally regardless of previous smoking status. Patients, and eventually staff, tolerated the smoking ban without significant negative effects. Patients relied very little on treatment modalities to alleviate nicotine withdrawal. Pre-ban apprehension by staff and patients dissipated with time after the smoking ban started.

Blueberry supplemented diet: effects on object recognition memory and nuclear factor-kappa B levels in aged rats.

It has been reported that an antioxidant-rich, blueberry-supplemented rat diet may retard brain aging in the rat. The present study determined whether such supplementation could prevent impaired object recognition memory and elevated levels of the oxidative stress-responsive protein, nuclear factor-kappa B (NF-kappaB) in aged Fischer-344 rats. Twelve aged rats had been fed a 2% blueberry supplemented diet for 4 months prior to testing. Eleven aged rats and twelve young rats had been fed a control diet. The rats were tested for object recognition memory on the visual paired comparison task. With a 1-h delay between training and testing, aged control diet rats performed no better than chance. Young rats and aged blueberry diet rats performed similarly and significantly better than the aged control diet group. Levels of NF-kappaB in five brain regions of the above subjects were determined by western blotting assays. In four regions, aged control diet rats had significantly higher average NF-kappaB levels than young animals on the control diet. In four regions, aged blueberry diet rats had significantly lower levels of NF-kappaB than aged control diet rats. Normalized NF-kappaB levels (averaged across regions and in several individual regions) correlated negatively and significantly with the object memory scores.