20230808-AI coding-1st round/359 – Lee 2014.txt

PMID: 24704083 PMCID: PMC4077337 DOI: 10.1016/j.neuropharm.2014.03.011
2. Materials and methods
2.1. Subjects
All experiments were conducted with approval from the Institutional Animal Care and Use Committee at the University of California, San Francisco. Sprague Dawley dams with litters containing male-only and female-only pups were obtained from Charles River Laboratories (Gilroy, CA). On postnatal day (P)7, animals were randomly assigned to control or treatment groups (Fig. 1). Following treatment, subjects were either killed and fixed for histology or cross fostered between dams. At P21, before reaching sexual maturity, each animal's sex was assessed and they were separated into groups by sex. Control and treatment animals were kept together in clean acrylic cages with bedding changed weekly and ad libitum access to food and water. Cages for both sexes were kept in the same room within the animal care facility with 12 h light–dark cycle and regulation of temperature (18–25 °C) and humidity (45–65%). At P30, they were housed in pairs with one treatment and one control animal per cage. All behavioral testing occurred during the light cycle between 0800 and 1700 h. Animals were food restricted for tasks involving object recognition. Access to food was limited to the light cycle in order to increase activity and object exploration during the testing period.
2.2. Anesthesia
Male and female subjects were separately anesthetized for a duration of four hours as we have previously described (Stratmann et al., 2009c). Briefly, isoflurane was delivered into the anesthetic chamber, and gas concentrations were continuously monitored. The isoflurane concentration was initially set to 4% (time = 0 min) and subsequently maintained at 1 Minimum Alveolar Concentration (MAC, the concentration required to prevent movement in 50% of subjects in response to a painful stimulus, Fig. 2). Every 15 min after induction, a supramaximal pain stimulus was produced by applying an alligator clamp to each rat's tail. Movement was defined as any gross movement other than breathing, and the percent of animals that moved in response to tail-clamping was calculated. Isoflurane concentration was then adjusted to maintain 50% response to the stimulus. Control animals were treated identically without tail-clamping or administration of anesthetic. Animals in the anesthesia chamber were kept on a warming blanket and the temperature was measured every 15 min using infrared thermometer, and the position and heating were adjusted to maintain normothermia.
2.3. Histology
Brains from male and female treatment and control groups (n = 10 per group) were assessed for acute neuronal death. Twelve hours after anesthesia, animals were anesthetized and transcardially perfused with cold 4% paraformaldehyde in phosphate-buffered saline and brains were removed, postfixed, and sunk in sucrose solution. They were then sliced into 60 micron-thick slices and every other slice was mounted and stained with FluoroJade C, a marker highly specific for neurodegeneration (FJC, 0.001%, Millipore, Billerica, MA). FJ-positive cells were counted using Nikon Eclipse 80i microscope under 20× magnification in each slice containing the structure of interest. Structures included in analysis were the anterodorsal (AD), anteroventral (AV), laterodorsal (LD), and anteromedial (AM) thalamic nuclei, as well as CA1–3 regions of the hippocampus and the dentate gyrus.

Because the sex of newborns rats is often ambiguous, genetic screening was used to confirm sex as described elsewhere (Miyajima et al., 2009). Briefly, DNA was isolated from tissue samples, and Sex-determining region Y (Sry, male-specific) and beta actin (autosomal) gene sequences were amplified by polymerase chain reaction (PCR) using Taq DNA polymerase (G-Biosciences, St. Louis, MO) and primers obtained from Eurofins MWG Operon (Huntsville, AL). After isolation of genomic DNA, PCR products were subjected to electrophoresis in 2% agarose gel, and males were identified by presence of two separate bands and females with a single band.

2.4. Object recognition tasks
Testing occurred similar to the paradigm used by others (Eacott and Norman, 2004, Langston and Wood, 2010). Male and female subjects were assessed using the same testing area and objects. Testing arenas and objects were wiped with 70% ethanol between subjects. Object recognition testing took place in two separate testing arenas, hereafter referred to as “contexts”, of identical size (61 cm square base, walls 50 cm high). The two were distinct in their appearance and texture to allow testing of context-specific memory. Context 1 had yellow walls and a base covered in wood-effect vinyl lining, while context 2 had black walls and a black plastic base. Visual cues were placed on three different walls within each context. Animals were introduced into the contexts facing the same direction and in the same location, and subjects were habituated to the contexts prior to testing. Each object was validated to avoid object bias. Investigation of an object was defined as sniffing or placing the nose within 1 cm of and oriented toward the object. Subjects were video recorded and reviewed by blinded observers to determine investigation times.

All subjects underwent the full series of testing in the order presented here with one trial per day. The subjects' order of testing also rotated each day so that the timing of behavioral testing was counterbalanced among subjects and groups. Testing began at postnatal day 38 (P38) with novel object recognition (Fig. 3). Subjects were assessed in their ability to recall a previously encountered object. A single trial was performed, and half of the subjects were tested in context 1 and the other half in context 2. During the “exposure”, the subject was placed into the context and explored two identical objects for four minutes. Following a two-minute delay, in the “test” phase, the animal was placed into the same context with one of the previous objects replaced with a novel object. The location (left or right) of the novel object within each context was counterbalanced among subjects. For each task, object investigation times during the initial exposure were compared, given possible confounding effects of varying investigation times on object recognition in the subsequent test phase.
Using object recognition as the premise, the tasks were then made increasingly complex. By using different objects and varying the locations and contexts in which they were presented, subjects were assessed in their ability to associate an object with a particular location, context, or combination of location and context. The arrangement used to assess each of these associative memory tasks is presented in Fig. 3.

In the final task of object–place–context recognition, control female subjects were identified as having increased object investigation during the exposure, thereby potentially conferring an advantage in subsequent object recognition. The following set of trials (Trials 3 and 4) were therefore performed while controlling for investigation times. Subjects were observed during the exposure with a goal of 15 s of investigation per object. Animals remained in the context for a minimum of two minutes and a maximum of five minutes to ensure adequate familiarization to the context. After the two-minute mark, if they reached the required investigation times, then they were removed. The test phase lasted four minutes and was recorded and later reviewed.

2.5. Social behavior and social recognition
Social interaction and recognition were assessed using a discrimination paradigm. In the “exposure” phase, the subject was presented with a caged stimulus animal alongside an empty cage for five minutes. This arrangement evaluates social interaction by determining whether subjects appropriately spend more time investigating the social target (Satomoto et al., 2009). After a sixty-minute delay, the subject was presented simultaneously with the same “familiar” stimulus animal and a novel animal for three minutes. Social recognition is demonstrated by decreased investigation of the familiar target relative to the novel one.

Same-sex juvenile conspecifics were used as stimulus animals. Male and female pups five weeks of age were housed individually one week prior to testing. Investigation was defined as any direct contact with the subject's nose or paws, as well as sniffing toward any part of the juvenile including the tail if it extended outside of the cage. Investigation of the empty cage was defined as sniffing or placing the nose within 1 cm of and oriented toward the cage, and excluded using the cage as a support during rearing.

2.6. Statistical analysis
Data were analyzed using Prism 6 Software for Mac OSX (GraphPad Software Inc., San Diego, CA). Data were assessed for normal distribution using the D'Agostino-Pearson test. Parametric tests were used for normally distributed data; otherwise, a nonparametric test was used. All comparisons used a two-tail test and a P value less than 0.05 was considered statistically significant.

Subjects were evaluated in their ability to recognize familiar stimuli, reflected by the relative time spent investigating two separate targets. For the final task (object–place–context recognition), times from Trials 1 and 2 were combined for analysis, and Trials 3 and 4 were assessed together. The ratio paired t-test was used to compare normally distributed data, and nonparametric data were analyzed with the Wilcoxon matched-pairs rank test. In addition, a “discrimination index” (DI) was calculated, representing the time spent investigating the novel target relative to the familiar target. To calculate DI, the time spent investigating the familiar target was subtracted from the time spent on the novel target, and this was divided by the total (eg. DI = (Novel − Familiar)/(Total Time)). DI provides a single value and therefore allows analysis by two-way ANOVA to compare effects of treatment or sex.

To identify and control for possible confounding effects of varying investigation times on subsequent object/animal recognition, the investigation times during the exposure phase were compared between the groups. These times were compared using one-way ANOVA for normally distributed data and Kruskal–Wallis test for nonparametric data. Bonferonni's post-test with multiple comparisons was used following one-way ANOVA, and Dunn's post-test was used with the Kruskal–Wallis test.

Two-way ANOVA was used to assess the effects of sex and treatment on neuronal death. Neuronal death for each brain region was compared using two-way ANOVA and Bonferroni post-test. The fold-increase in neuroapoptosis was determined for each structure by dividing the total FJ-positive cells of each treatment animal (n = 20) by the average number of FJ-positive cells per structure for the whole control group (n = 20).