GABAA Receptor-Mediated Acceleration of Aging-Associated Memory Decline in APP/PS1 Mice and Its Pharmacological Treatment by Picrotoxin

Advanced age and mutations in the genes encoding amyloid precursor protein (APP) and presenilin (PS1) are two serious risk factors for Alzheimer's disease (AD). Finding common pathogenic changes originating from these risks may lead to a new therapeutic strategy. We observed a decline in memory performance and reduction in hippocampal long-term potentiation (LTP) in both mature adult (9–15 months) transgenic APP/PS1 mice and old (19–25 months) non-transgenic (nonTg) mice. By contrast, in the presence of bicuculline, a GABAA receptor antagonist, LTP in adult APP/PS1 mice and old nonTg mice was larger than that in adult nonTg mice. The increased LTP levels in bicuculline-treated slices suggested that GABAA receptor-mediated inhibition in adult APP/PS1 and old nonTg mice was upregulated. Assuming that enhanced inhibition of LTP mediates memory decline in APP/PS1 mice, we rescued memory deficits in adult APP/PS1 mice by treating them with another GABAA receptor antagonist, picrotoxin (PTX), at a non-epileptic dose for 10 days. Among the saline vehicle-treated groups, substantially higher levels of synaptic proteins such as GABAA receptor α1 subunit, PSD95, and NR2B were observed in APP/PS1 mice than in nonTg control mice. This difference was insignificant among PTX-treated groups, suggesting that memory decline in APP/PS1 mice may result from changes in synaptic protein levels through homeostatic mechanisms. Several independent studies reported previously in aged rodents both an increased level of GABAA receptor α1 subunit and improvement of cognitive functions by long term GABAA receptor antagonist treatment. Therefore, reduced LTP linked to enhanced GABAA receptor-mediated inhibition may be triggered by aging and may be accelerated by familial AD-linked gene products like Aβ and mutant PS1, leading to cognitive decline that is pharmacologically treatable at least at this stage of disease progression in mice

Effects of Arabidopsis Ku80 deletion on the integration of the left border of T-DNA into plant chromosomal DNA via Agrobacterium tumefaciens

T-DNA integration into plant chromosomal DNA via Agrobacterium tumefaciens can be achieved by exploiting the double-strand break repair system of the host’s DNA. However, the detailed mechanism of T-DNA integration remains unclear. Here, a sequence analysis of the junction sequences of T-DNA and chromosomal DNA was performed to assess the mechanism of T-DNA integration. T-DNA was introduced into Arabidopsis wild-type and NHEJ-deficient ku80-mutant plants using the floral dip method; the junctions of the left border (LB) of T-DNA were subsequently analyzed by adapter PCR. The most frequent junction of the LB of T-DNA to chromosomal DNA (LB-to-ChrDNA) was of the filler DNA type in both lines. The lengths of direct or inverted repeat sequences within or around the filler DNA sequence were longer in the ku80 mutant. In addition, the frequency of T-DNA integration near the transcription start site was significantly higher in the ku80 mutant. Our observations suggest that the presence of the Ku80 protein affects the location of the integration of T-DNA and the pattern of the formation of repeat sequences within or around the filler DNA during LB integration into chromosomal DNA

Contribution of Nucleus Accumbens Core (AcbC) to Behavior Control during a Learned Resting Period: Introduction of a Novel Task and Lesion Experiments

<div><p>In recent years, the study of resting state neural activity has received much attention. To better understand the roles of different brain regions in the regulation of behavioral activity in an arousing or a resting period, we developed a novel behavioral paradigm (8-arm food-foraging task; 8-arm FFT) using the radial 8-arm maze and examined how AcbC lesions affect behavioral execution and learning. Repetitive training on the 8-arm FFT facilitated motivation of normal rats to run quickly to the arm tips and to the center platform before the last-reward collection. Importantly, just after this point and before confirmation of no reward at the next arm traverse, locomotor activity decreased. This indicates that well-trained rats can predict the absence of the reward at the end of food seeking and then start another behavior, namely planned resting. Lesions of the AcbC <i>after</i> training selectively impaired this reduction of locomotor activity after the last-reward collection without changing activity levels before the last-reward collection. Analysis of arm-selection patterns in the lesioned animals suggests little influence of the lesion in the ability to predict the reward absence. AcbC lesions did not change exploratory locomotor activity in an open-field test in which there were no rewards. This suggests that the AcbC controls the activity level of planned resting behavior shaped by the 8-arm FFT. Rats receiving training after AcbC lesioning showed a reduction in motivation for reward seeking. Thus, the AcbC also plays important roles not only in controlling the activity level after the last-reward collection but also in motivational learning for setting the activity level of reward-seeking behavior.</p></div

Schematic representation of hypothesized behavioral control according to changes in reward condition.

<p>From the presented results, we propose that the activity level in planned resting behavior is determined by intensity level of the global activity controller and the AcbC. The former regulates the level of platform-approaching behavior and behavior during the reward-unavailable period after the last-reward collection (LRC). The latter selectively controls the resting level in the reward-unavailable period after LRC. Height of gray highlighted areas represents intensity. <b>A</b>: In the early phase of 8-arm FFT training, the output intensities of the global activity controller and AcbC are relatively low. Thus, behavioral activity level (speed) of a platform approach before the LRC is relatively low, and the level of planned resting after LRC is not enough to inhibit redundant actions during the reward-unavailable period. <b>B</b>: After the repetitive training on the 8-arm FFT, the output intensities of global controller and AcbC are higher than those in early phase of training, contributing to the formation of a temporal behavior pattern that reflects the change in the reward situation. <b>C</b>: Lesioning of the AcbC after training enhances the activity level of planned resting after the LRC, because AcbC damage causes disinhibition. <b>D</b>: By contrast, repetitive training after lesion of AcbC fails to adjust the output level of the global controller to a higher state. This is because AcbC-mediated inhibition of (redundant) actions during the reward-unavailable period is required for learning to enhance the activity level of global controller.</p

Effect of AcbC lesions on the execution of optimized behavior in the 8-arm FFT.

<p>AcbC (n = 6 rats) or sham control lesions (n = 6) were made in well-trained rats. <b>A</b>: Averaged tip-approaching (TA) speeds before the last-reward collection (LRC) did not differ in well-trained AcbC-lesioned rats compared to sham controls (t = 0.67, <i>P</i>>0.05, unpaired <i>t</i>-test). <b>B</b>: Averaged platform-approaching (PA) speeds before LRC in AcbC-lesioned rats also did not differ (t = 0.85, <i>P</i>>0.05, unpaired <i>t</i>-test). <b>C</b>: For the one min after the last reward was eaten (LRC), AcbC-lesioned rats had more re-entry errors compared to sham controls (t = 3.76, <i>P</i><0.05, unpaired <i>t</i>-test). <b>D</b>: Rats with AcbC lesions showed increased locomotion activity after LRC (t = 4.47, <i>P</i><0.05, unpaired <i>t</i>-test). <b>E</b>: Mean PA speed just before (7th arm visit) and just after (8th arm visit) LRC. In contrast to sham-lesioned rats, AcbC-lesioned rats did not significantly decrease their PA speed just after LRC (<i>F</i>_1,5 = 28.27, <i>P</i><0.05, two-way ANOVA; AcbC-lesions, <i>P</i>>0.05; sham-lesions, <i>P</i><0.05, Sidak's multiple comparison test). *<i>P</i><0.05 compared to sham controls; #<i>P</i><0.05 compared to 7th arm visit. Error bars denote SEM. LRC, the last-reward collection; GO, gate-open; TA, tip-approaching; PA, platform-approaching.</p

Introduction of the 8-arm food foraging task and performance of normal adult rats.

<p><b>A</b>: Schematic design of the 8-arm food foraging task (8-arm FFT). Horizontal bar shows the sequence of task phases and critical events during a trial. Each rat was placed on the platform with all arm gates closed (before-GO period). Two to three minutes later, all gates were opened (GO), allowing free access to the tip of each arm, where the reward was located in a food cup. Five minutes after the last-reward collection (LRC), the rat was removed and placed in its home cage. Bottom row of diagrams in (A) shows movement traces during the different phases. Each of the solid, irregular lines shows a typical example of well-trained rat movements (41st–45th trials). Repeated training produced a pattern of reliable and low-cost behavior, which was characterized by automatic-appearing sequential arm selection before LRC. After the LRC (reward-<u>un</u>available period), random patterns were common, with much slower running speeds compared to before the LRC. <b>B</b>: Timing of changes in mean tip-approaching (TA, black filled bars) and platform-approaching speed (PA, open bars) of well-trained rats (41st–45th trials). The platform-approaching speed at the 8th approach (just after LRC) was significantly slower than that of the 7th approach (one-way ANOVA, <i>F</i>_11,156 = 100.8, <i>P</i><0.05; Dunn-Sidak test, * <i>P</i><0.05). Similar to platform approaching, a significant slowing of tip-approaching speeds occurred between the 8th and 9th approaches (<i>P</i><0.05). Dashed line shows at which point the LRC occurs. <b>C</b>: Change in the number of arm re-entry errors before LRC (reward-available period). <b>D</b>: Change in average tip-approaching and platform-approaching speeds before LRC. <b>E</b>: Change in the number of arm re-entry errors for one minute after LRC. <b>F</b>: Change in traveling distance for one minute after LRC. The various experimental groups are explained in detail in the Supplementary Methods. Error bars denote SEM.</p

Histological analysis of AcbC-lesioned rats.

<p>Extent of ibotenic acid AcbC lesions in rats for two sets of experiments: before the start of repetitive training on the 8-arm FFT (n = 6) and after repetitive training (n = 6) <b>A</b>: Representative photomicrographs of Nissl-stained coronal sections showing the AcbC in sham control and lesioned rats. Bidirectional reduction of neuronal components is present around the ibotenic acid injection site (ii). Sham control rats showed very little destruction around the AcbC (i). Higher magnification images in iii and iv. AcbC, nucleus accumbens core; AcbS, nucleus accumbens shell; ac, anterior commissure; LV, lateral ventricle. <b>B</b>: Composite drawings of the extent of lesioned areas (filled gray) in standardized coronal drawings as determined in all behaviorally assessed rats. Darker gray areas indicate overlap of lesions among rats. Numbers represent the rostrocaudal distance (mm) from bregma. Left column of sections shows the size and location of the lesioned areas in rats receiving an AcbC lesion <i>after</i> training (n = 6), whereas the right column shows the size and location of the lesioned areas in rats receiving an AcbC lesion <i>before</i> training (n = 6). Plates were adapted from the atlas of Paxinos and Watson <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095941#pone.0095941-Paxinos1" target="_blank">[29]</a>.</p

Activity level after the LRC correlates with platform-approaching speed preceding the LRC.

<p>Platform-approaching speeds before the LRC (reward-available period) and distances traveled after the LRC (reward-<u>un</u>available period) were analyzed using data obtained during the 31st-45th trials on the 8-arm FFT. Performance of AcbC-lesioned rats (n = 12) and intact, unlesioned rats (n = 30) were compared. Both groups showed statistically significant correlations between platform-approaching speeds before the LRC and activity level after the LRC (distances traveled) (intact group: r = +0.44, <i>P</i><0.05; AcbC-lesioned group: r = +0.68, <i>P</i><0.05), and the two regression lines were parallel (slopes; <i>P</i>>0.05). However, the regression line for the AcbC-lesion group shifted toward the right compared to the one for the intact group. Filled square and triangle symbols are average values for each rat. LRC, last-reward collection; PA, platform-approaching.</p