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BackgroundAt present, several positron emission tomography (PET) tracers are in use for imaging P-glycoprotein (P-gp) function in man. At baseline, substrate tracers such as R-[11C]verapamil display low brain concentrations with a distribution volume of around 1. [11C]phenytoin is supposed to be a weaker P-gp substrate, which may lead to higher brain concentrations at baseline. This could facilitate assessment of P-gp function when P-gp is upregulated. The purpose of this study was to synthesize [11C]phenytoin and to characterize its properties as a P-gp tracer.Methods[11C]CO was used to synthesize [11C]phenytoin by rhodium-mediated carbonylation. Metabolism and, using PET, brain pharmacokinetics of [11C]phenytoin were studied in rats. Effects of P-gp function on [11C]phenytoin uptake were assessed using predosing with tariquidar.Results[11C]phenytoin was synthesized via [11C]CO in an overall decay-corrected yield of 22 ± 4%. At 45 min after administration, 19% and 83% of radioactivity represented intact [11C]phenytoin in the plasma and brain, respectively. Compared with baseline, tariquidar predosing resulted in a 45% increase in the cerebral distribution volume of [11C]phenytoin.ConclusionsUsing [11C]CO, the radiosynthesis of [11C]phenytoin could be improved. [11C]phenytoin appeared to be a rather weak P-gp substrate.

The aim of the present study was to investigate if flumazenil blood-brain barrier transport and binding to the benzodiazepine site on the g-aminobutyric acid A (GABA A ) receptor complex is altered in an experimental model of epilepsy and subsequently to study if changes in P-glycoprotein (P-gp)-mediated efflux of flumazenil at the blood-brain barrier may confound interpretation of 11 C-flumazenil PET in epilepsy. Methods: The transport of flumazenil across the blood-brain barrier and the binding to the benzodiazepine site on the GABA A receptors in 5 different brain regions was studied and compared between controls and kainate-treated rats, a model of temporal lobe epilepsy, with and without tariquidar pretreatment. In total, 29 rats underwent 2 consecutive 11 C-flumazenil PET scans, each one lasting 30 min. The tracer was mixed with different amounts of isotopically unmodified flumazenil (4, 20, 100, or 400 mg) to cover a wide range of receptor occupancies during the scan. Before the second scan, the rats were pretreated with a 3 or 15 mg/kg dose of the P-gp inhibitor tariquidar. The second scan was then obtained according to the same protocol as the first scan. Results: GABA A receptor density, B max , was estimated as 44 6 2 ngÁmL 21 in the hippocampus and as 33 6 2 ngÁmL 21 in the cerebellum, with intermediate values in the occipital cortex, parietal cortex, and caudate putamen. B max was decreased by 12% in kainate-treated rats, compared with controls. The radiotracer equilibrium dissociation constant, K D , was similar in both rat groups and all brain regions and was estimated as 5.9 6 0.9 ngÁmL 21 . There was no difference in flumazenil transport across the blood-brain barrier between control and kainate-treated rats, and the effect of tariquidar treatment was similar in both rat groups. Tariquidar treatment also decreased flumazenil transport out of the brain by 73%, increased the volume of distribution in the brain by 24%, and did not influence B max or K D , compared with baseline . Conclusion: B max was decreased in kainate-treated rats, compared with controls, but no alteration in the bloodbrain barrier transport of flumazenil was observed. P-gp inhibition by tariquidar treatment increased brain concentrations of flumazenil in both groups, but B max estimates were not influenced, suggesting that 11 C-flumazenil scanning is not confounded by alterations in P-gp function.

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This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal's behavioral profile can be investigated in one single test.The apparatus consists of a box, representing the 'protected' area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the 'unprotected' area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related-and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals' adaptive capacities.Due to testing order effects in a behavioral test battery, naïve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

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Alcohol use disorder (AUD) is characterized by excessive and persistent alcohol use, despite adverse consequences. AUD often originates during adolescence, as do other substance use disorders. However, despite periods of excessive alcohol intake, many adolescents reduce their alcohol use by early adulthood. Brain development, social context, personality traits, and genetic makeup are thought to play an important role in these age-dependent fluctuations in alcohol use. However, studies that directly investigate age-related differences in the effects of alcohol exposure on brain and behavior are sparse. Therefore, to better understand the relationship between adolescent alcohol consumption and AUD-like behavior, this study compared the degree of control over alcohol seeking in rats that differed in terms of age of onset of alcohol drinking and in their level of alcohol consumption. We hypothesized that control over alcohol seeking is more prominent in adolescent-onset rats than in adult-onset rats, and that control over alcohol seeking is related to the consumed amount of alcohol. To test this hypothesis, alcohol seeking in the presence of a conditioned aversive stimulus was assessed after 2 months of intermittent alcohol access (IAA) in rats that consumed alcohol from postnatal day 42 (adolescence) or day 77 (adulthood). The rats were subdivided into low (LD), medium (MD), or high (HD) alcohol drinking rats, in order to assess the impact of the extent of alcohol intake on control over alcohol seeking. The adolescent-onset animals consumed slightly, but significantly less alcohol compared to the adult-onset rats. In adult-onset rats, we found that conditioned suppression of alcohol seeking, i.e., reduction of alcohol seeking by presentation of a conditioned aversive stimulus, was most pronounced in LD. By contrast, in the adolescent-onset rats, MD and HD showed increased alcohol seeking compared to LD, which was suppressed by conditioned aversive stimuli. Taken together, these findings reveal a complex relationship between the age of onset and level of alcohol intake with control over alcohol seeking, whereby adolescent rats consume less alcohol than adults. In adult rats, control over alcohol seeking is negatively related to preceding levels of alcohol intake. By contrast, adolescent rats appear to retain control over alcohol seeking, even after a history of high levels of alcohol intake.

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