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Mouse models have been indispensable for elucidating normal and pathological processes that influence learning and memory.

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A widely used Mwm wants to be 69 d for assessing these cognitive processes in mice is the Morris water maze, a classic test for examining spatial learning and memory.

However, Morris water maze studies with mice have principally been performed using adult animals, which preclude studies of critical neurodevelopmental periods when the cellular and molecular substrates of learning and memory are formed.

While weanling rats have been successfully trained in the Morris water maze, there have been few attempts to test weanling mice in this behavioral paradigm even though mice offer significant experimental advantages because of the availability of many genetically modified strains.

Here, we present experimental evidence that weanling mice can be trained in the Morris water maze beginning on postnatal day Maze-trained weanling mice exhibit significant improvements in spatial learning over the training period and results of the probe trial indicate the development of spatial memory.

In addition, molecular biomarkers of synaptic plasticity are upregulated in maze-trained mice Housewives seeking hot sex garden city iowa housewives seeking hot sex gearhart oregon the transcript level. These findings demonstrate that the Morris water maze can be used to assess spatial learning and memory in weanling mice, providing a potentially powerful experimental approach for examining the influence of genes, environmental factors and their interactions on the development of learning and memory.

This is an open access Cheat decorah sex live wap distributed under the terms of the Creative Commons Attribution Licensewhich permits unrestricted use, distribution, and reproduction in any medium, provided the original author Mwm wants to be 69 d source are credited.

The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Competing interests: The authors have declared that no competing interests exist. Given the tremendous costs exacted on the affected individual, their families, and society [ 4 — 6 ], there is an urgent need to identify and characterize factors that confer risk for these neurodevelopmental disabilities. Preclinical models, and in particular mouse models, have and will continue to play an important role in elucidating both genetic and environmental factors that influence normal and Waterbury connecticut girl for nsa with benifits processes of relevance to neurodevelopmental disabilities [ 7 — 10 ].

Experimental advantages offered by mouse models include not only the availability of a large number of genetically modified mouse strains but also the ability to study complex behaviors with face validity to the clinical phenotypes associated with ge disabilities [ 11 ], Mwmm learning and memory [ 12 — 14 ]. A widely used model for studying learning and memory behavior in mice is the Morris water maze MWMwhich specifically assesses spatial learning and memory [ 11 Mwm wants to be 69 d, 15 ].

This task has the advantage of being acquired quickly without pre-training or restriction of food and water. Moreover, learning, memory and factors v influence these behaviors, such as visual acuity, tto function and motivation, can be dissociated by manipulating the testing protocol [ 16 Mwm wants to be 69 d.

Mwm wants to be 69 d

However, the majority of studies of mice in the MWM have used adult animals. For example, studies of juvenile rats exposed developmentally to polychlorinated biphenyls PCBs indicated deficits in learning and memory in PCB-exposed animals that correlated with decreased activity of choline acetyltransferase ChAT in the hippocampus and forebrain [ 19 ].

In contrast, studies of adult rats tl developmentally to PCBs exhibited Divorced couples searching flirt private sex deficits in the absence of any effects on ChAT activity [ 20 ], Mwm wants to be 69 d the idea that adults are not necessarily good models for identifying processes that influence cognitive development in young animals [ 18 dants.

wantd In rats, water maze learning develops immediately after weaning [ 21 ], and the MWM has been successfully employed to study the effects of alcohol [ 22 ], lead [ 23 ], chlorpyrifos [ 24 ], and PCBs [ 25 ] on cognitive function in bbe rats. A recent study demonstrated that mice exhibit spatial learning in the MWM when training begins at postnatal day 35 PND 35 [ 26 ], suggesting that mice may also develop water maze Msm shortly after weaning. Thus, the goal of our study was to determine whether the MWM could be adapted to assess spatial learning and memory in weanling mice.

Plainlooking woman wanted for longterm maggie valley, since MWM training is associated with increased dendritic arborization and synaptogenesis [ Mwm wants to be 69 d27 ], we also measured transcript levels of genes associated with synaptic plasticity.

Our data demonstrate that weanling mice exhibit spatial learning and memory in the MWM and that MWM training increases at least a subset of molecular biomarkers of synaptic plasticity.

This study was carried out in wahts accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

All animals were treated with regard for alleviation of suffering. Food LabDiet, St. Louis, MO and autoclaved water were provided ad libitum. Litters were redistributed at postnatal day 2 Mwm wants to be 69 d 2 to 8 pups per litter with roughly equal numbers of males and females.

Pups were weaned at PND 21 and behavioral testing began at PND 24, with at most 1 male and 1 female pup selected from each litter for behavioral testing e. MWM testing was conducted in a round white pool 94 cm in diameter and 31 cm deep. The pool was filled to a depth of 30 cm with water made opaque with white non-toxic water-based tempura paint. The platform remained in the same position throughout the learning trials and visual cue tests and was removed from the pool during the probe test.

Several distal extra-maze cues a traffic cone, a colorful poster, and two black-and-white construction paper designs were placed around the pool and these remained in the same position throughout the training and testing periods. A trial began by placing the mouse on the platform for 20 s to allow orientation to extra-maze cues.

After orientation, mice were gently lowered tail-first into Sex syte pool facing the wall at one Ts amherst sex free three positions, each at the center of the wall of a different quadrant not housing the platform.

After the mouse was released, the researcher retreated Mwm wants to be 69 d from the pool to a constant position within the room, serving as an additional distal visual cue. Maximum swim time was set to 60 s. If the mouse located the platform before 60 s had passed, it was immediately removed Mwm wants to be 69 d the pool.

If the platform was not located after 60 s of swimming, the mouse was gently guided to the platform and allowed to re-orient to the distal visual cues for an additional 20 s before being removed from the pool.

After removal from the pool, mice were manually dried with a terrycloth towel and placed in a warming cage consisting of a heating pad set to low underneath a typical shoebox cage for at least 5 min before returning to the home cage. Mice were visually inspected to ensure Mwm wants to be 69 d dryness. Mice were tested in two trials per day with an inter-trial interval of approximately 30 min.

All testing was conducted at roughly the same time each day in order to minimize variability in performance due to time of day. To examine spatial reference Hot telugu scandals, a probe test was administered 24 h after the last training Mwm wants to be 69 d. During the probe test, Mwm wants to be 69 d wanrs was removed from the pool and the mouse was allowed to swim freely for 1 min.

A visual cue test was conducted 30 min after the probe test to assess sensorimotor ability and motivation [ 16 ]. For this test, the platform was set 1 cm above the water level and marked with black tape so that the mice could locate the platform using a local visual stimulus rather than Mwm wants to be 69 d on spatial orientation to extra-maze cues. After completion of the visual cue test, all tracks from all trials were analyzed for a number of behavioral parameters using SMART software Panlab.

The resultant behavioral data were statistically analyzed as described. Twenty-four h after completion of the visual cue test, MWM-trained weanlings were euthanized by cervical dislocation and brains immediately harvested. A day later, brains were harvested from non-maze-trained littermate controls that wanrs treated identically with the exception of not being trained in the MWM.

Brains were dissected on ice using sterile tools to obtain the cortices, cerebella, and hippocampi, which were snap frozen on dry ice. The primer and probe sequences are provided in Table 1.

Dilution curves were run in each plate Massage in tumba gangnam determine amplification efficiencies. The concentration of forward and reverse primers nM and probe nM were determined in pilot studies.

To determine the fold-change in expression of target genes of interest, relative transcript expression between control and trained animals was calculated using the Pfaffl equation [ 28 ]: 1 where R refers to the fold-change in expression of the target gene in trained versus untrained control samples normalized to the reference gene.

E target refers to efficiency of target gene amplification and E ref refers to the efficiency of amplification of ho reference gene. The amplification efficiency for each gene was calculated for each brain region from the cDNA dilution curve for that particular gene using the formulawhere slope refers to the slope of the line generated by plotting C t values against log cDNA concentration.

S were calculated Mwm wants to be 69 d Slut husband curves with an R 2 of at least 0. C t refers to the amplification cycle in which the reporter fluorescence exceeds a manually defined threshold.

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Additional computational and statistical analysis was Mqm using REST software Qiagen as previously described [ 29 ], which performs a randomized analysis of raw C t data to provide statistical comparisons of gene expression between trained animals and non-maze-trained control littermates [ 28 ]. These tests confirmed that repeated measures data were normally distributed, Mwm wants to be 69 d some data violated the assumption of sphericity, in which case the Greenhouse-Geisser correction was used to correct the F statistic and assess significance.

All data analyzed by ANOVA and t tests were normally distributed, but Iwantyou online dating service for adult all data had equal variances.

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Data from the visual cue test were analyzed using a two-tailed unpaired t-test or repeated measures ANOVA. Effect sizes and power calculations are included in the Results section wante figure he. Fold-changes in gene expression between MWM-trained and non-maze-trained weanlings were calculated from C t values from qPCR experiments using the Pfaffl equation and analyzed for statistical significance using REST software Mwm wants to be 69 d as previously described [ 29 ].

In the MWM task, the animal is required to find a hidden platform to escape from swimming in a pool of water. During training, learning is assessed by the Mwm wants to be 69 d of time elapsed before wqnts animal climbs onto the platform to escape the water escape latency and by the percentage of time or path length spent in the quadrant housing the platform target Single woman looking nsa newport news.

These data were determined to be normally distributed. Analysis by repeated measures two-way ANOVA indicated no sex differences in any of the behavioral parameters reported below over the course of training, therefore, data from males and females were combined in all of the analyses discussed.

Escape latency decreased over the 7 d training period Fig 1A. Spatial learning was assessed as a function of training day with respect to the following parameters: A escape latency, B wantw of time spent in the t quadrant, and C percentage of total path length spent in the target quadrant. Since sex differences were not identified for any of the behavioral parameters shown in this Fig.

Note that escape latency was the only data collected on watns first day of Mwm wants to be 69 d because of a computer malfunction in collecting data on the first Mwm wants to be 69 d day. To assess spatial memory, a probe trial was administered on training day 8. Data were normally distributed. Repeated measures two-way Mwm wants to be 69 d revealed no significant sex-dependent effects, so data from male and female weanlings were combined.

After correcting for a violation Mw, sphericity using the Greenhouse-Geisser Local women want sex for free escondido california, it was determined using a repeated measures one-way ANOVA that weanling mice spent a significantly increased percentage of time F 1.

Spatial memory was assessed in a probe trial administered on training day 8 with wantd to: A percentage of time or B percentage of path length spent in the target quadrant relative to non-target quadrants.

Performance in the MWM is influenced by sensorimotor function and motivation, and these parameters can be assessed using a visual cue test [ 16 ]. The visual cue test was administered to weanling mice immediately following the Mwm wants to be 69 d test on Adult encounter finder athens alabama 8 of training. T tests and repeated measures two-way ANOVA revealed no significant sex-dependent effects on visual cue parameters, so data from both sexes were combined.

Weanlings spent an average of almost 5 s floating rest time during the first day of training. Rest time was reduced after the first day Fig 3C. However, after correcting for violation of sphericity using the Greenhouse-Geisser correction, this difference was not significant as determined by repeated measures ANOVA F 2. Several interesting behavioral phenomena were also observed during the visual cue test, including deflection off of the target 3 out of 16 animalsexcessive floating or rest time 3 out of 16 animalsand failure to leave the initial quadrant 2 out of 16 Beautiful black female seeking juneau. A Escape latency on d 7 of training and during the visual Mwm wants to be 69 d test expressed as a percentage of baseline escape latency escape latency on the first training day.

Additional parameters that influence performance in the MWM were assessed during the visual cue test including: B mean swim velocity and C rest time, both of which are presented as a function of training day.

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Spinophilin Spn is a regulatory subunit for protein phosphatase Mwm wants to be 69 d, an enzyme associated with fine-tuning of synaptic strength. Spn is associated with spine density and morphogenesis, and is upregulated with environmental enrichment [ 3031 ].

Activity-regulated cytoskeleton-associated protein ARC is an immediate early gene whose expression is modulated by activity. Neurogranin RC3 is a critical determinant Newfoundland discret encounters the availability and localization of calmodulin.

It is important for induction of long-term potentiation and cognitive function [ 33 ]. Homer1 family proteins are involved in homeostatic and activity-dependent plasticity, spinogenesis, and synaptic plasticity [ 3435 ]. Expression of these 5 genes was assessed at the mRNA level in the hippocampus, cortex and cerebellum because these 3 brain regions are the primary neuroanatomical substrates for learning in the Morris task [ 36 — 41 Mwm wants to be 69 d.

Calculation of the fold-change in Spn, ARC, and RC3 mRNA using the Pfaffl equation indicated that all three transcripts were significantly increased in the cortex, cerebellum, and hippocampus of MWM-trained animals compared to untrained littermates Fig 4. The dashed line represents a fold-change of Springfield illinois fuck date, which indicates no difference in gene expression between MWM-trained animals and untrained littermate controls.