Age-Related Differences in Alcohol Intake and Control Over Alcohol Seeking in Rats.

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.

Effects of light regime and substrain on behavioral profiles of male C57BL/6 mice in three tests of unconditioned anxiety.

Substrains of the C57BL/6 inbred mouse are widely used in genetic, behavioral and physiological research, as well as models for human disease. Throughout, the choice of the respective substrain can have a large influence on experimental results. Likewise, the conditions under which experiments are performed, such as the light regime, can significantly affect the outcome of an experiment, especially when aiming at experimental behavior. Here, two commonly used mouse substrains, C57BL/6JOlaHsd and C57BL/6NCrl, were housed under either a conventional or a reverse light regime and were tested in either the light phase or the dark phase, respectively. All animals were exposed to three unconditioned anxiety-related behavior set-ups: the modified Hole Board test, the light-dark box and the elevated plus maze. Significant substrain and light regime effects were found in all three behavioral tests, with some of the latter being substrain and test specific. This signifies the importance of the choice of substrain used in for example, a mouse knockout experiment studying behavior, also in relation to light regime under which the animals are tested.

The modified hole board--measuring behavior, cognition and social interaction in mice and rats.

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.

[11C]quinidine and [11C]laniquidar PET imaging in a chronic rodent epilepsy model: impact of epilepsy and drug-responsiveness.

INTRODUCTION: To analyse the impact of both epilepsy and pharmacological modulation of P-glycoprotein on brain uptake and kinetics of positron emission tomography (PET) radiotracers [(11)C]quinidine and [(11)C]laniquidar. METHODS: Metabolism and brain kinetics of both [(11)C]quinidine and [(11)C]laniquidar were assessed in naive rats, electrode-implanted control rats, and rats with spontaneous recurrent seizures. The latter group was further classified according to their response to the antiepileptic drug phenobarbital into "responders" and "non-responders". Additional experiments were performed following pre-treatment with the P-glycoprotein modulator tariquidar. RESULTS: [(11)C]quinidine was metabolized rapidly, whereas [(11)C]laniquidar was more stable. Brain concentrations of both radiotracers remained at relatively low levels at baseline conditions. Tariquidar pre-treatment resulted in significant increases of [(11)C]quinidine and [(11)C]laniquidar brain concentrations. In the epileptic subgroup "non-responders", brain uptake of [(11)C]quinidine in selected brain regions reached higher levels than in electrode-implanted control rats. However, the relative response to tariquidar did not differ between groups with full blockade of P-glycoprotein by 15 mg/kg of tariquidar. For [(11)C]laniquidar differences between epileptic and control animals were only evident at baseline conditions but not after tariquidar pretreatment. CONCLUSIONS: We confirmed that both [(11)C]quinidine and [(11)C]laniquidar are P-glycoprotein substrates. At full P-gp blockade, tariquidar pre-treatment only demonstrated slight differences for [(11)C]quinidine between drug-resistant and drug-sensitive animals.

Altered GABAA receptor density and unaltered blood-brain barrier transport in a kainate model of epilepsy: an in vivo study using 11C-flumazenil and PET.

UNLABELLED: The aim of the present study was to investigate if flumazenil blood-brain barrier transport and binding to the benzodiazepine site on the γ-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 µg) 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 ± 2 ng x mL(-1) in the hippocampus and as 33 ± 2 ng x mL(-1) 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 ± 0.9 ng x mL(-1). 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 blood-brain 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.

[11C]phenytoin revisited: synthesis by [11C]CO carbonylation and first evaluation as a P-gp tracer in rats.

BACKGROUND: At 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. CONCLUSIONS: Using [11C]CO, the radiosynthesis of [11C]phenytoin could be improved. [11C]phenytoin appeared to be a rather weak P-gp substrate.

Altered N-methyl-D-aspartate receptor function in reelin heterozygous mice: male-female differences and comparison with dopaminergic activity.

The aim of this study was to investigate the in vivo relationship between reelin and NMDA receptor function in schizophrenia. We assessed the effect of reelin deficiency in behavioral models of aspects of this illness, NMDA receptor subunit levels, and NMDA receptor, dopamine D2 receptor, and dopamine transporter density. Male, but not female, reelin heterozygous mice showed significantly enhanced MK-801-induced locomotor hyperactivity compared to wildtype controls (7.4-fold vs. 5.2-fold effect of MK-801 over saline, respectively) but there were no genotype differences in the response to amphetamine. Both male and female reelin heterozygous mice showed enhanced effects of MK-801 on startle, but not prepulse inhibition (PPI) of startle. There were no group differences in the effect of apomorphine on startle or PPI. The levels of NMDA receptor subunits were not altered in the striatum. In the frontal cortex, male and female reelin heterozygous mice showed significant up-regulation of NR1 subunits, but down-regulation of NR2C subunits, which was associated with significantly elevated NR1/NR2A and NR1/NR2C ratios. However, there were no differences in [³H]MK-801 binding density in the nucleus accumbens or caudate nucleus, nor in the density of [³H]YM-09151 or [³H]GBR12935 in these brain regions. The enhanced effects of MK-801 in reelin heterozygous mice in this study could be reflective of the role of reelin deficiency in schizophrenia. This genotype effect was male-specific for locomotor hyperactivity, a model of psychosis, but was seen in male and female mice for startle, which could be an indication of changes in anxiety. Changes in NMDA receptor subunit levels and ratios were also seen in both male and female mice. These results suggest that the role of reelin deficiency in schizophrenia may be particularly mediated by altered NMDA receptor responses, with some of these effects being strictly sex-specific.