Novel Object Recognition Test

The behavioral test battery consisted of five memory tests performed in the following order: Y-maze forced alternation, novel object recognition, Morris water maze, radial arm water maze and Y-maze spontaneous alternation (Fig 1). Tests were conducted in the order of increasing invasiveness, with the exception of the Y-maze spontaneous alternation test. This test was conducted at the end of the test battery to minimize interference with the forced alternation test at the beginning of the study. Mice had multiple days of resting time between tests to decrease carryover effects from prior tests. The order of tests in which mice were tested was the same for each mouse; each mouse was tested once per test. Black and white cues were placed at the walls around the testing area for all tests except the novel object recognition trials. Cues were changed after the forced alternation test and remained the same for the remaining tasks. All test trials were video-recorded, tracked, and analyzed with ANY-maze^™ tracking software (version 499g Beta). Locomotor activity data for each test are summarized in Table 1. Mice were habituated to the testing room for 30 min at the beginning of each test day. All tests in the battery were conducted by the same experimenter. During the test trials, the experimenter was separated from the testing area by a curtain. All experiments comparing 129S6/Tg2576 to wild type mice were blinded. Memory tests are described in the order the tests were conducted.

The effects of IVIg treatment on memory and anxiety-like behavior in 3xTg-AD and NonTg mice were evaluated using a series of cognitive tests. Behavioral tests were performed during the 2 weeks preceding sacrifice with a recovery time of at least 48 hours between every task and a 24-hour delay after the last IVIg injection to reduce stress. The protocols used for all behavioral testing were based on pilot studies previously performed in our colony of 3xTg-AD mice
[18 (link),19 (link)].
The novel object recognition (NOR) test has been developed to study learning and memory in rodents and is based on their spontaneous tendency to have more interactions with a novel than a familiar object
[18 (link)]. During the familiarization period, the mouse was placed in a standard cage (29.2 cm × 19 cm × 12.7 cm) containing two identical objects for 5 minutes and returned quickly to its housing cage. Recognition memory was tested 1 hour later by exposing the animal to one familiar and one novel object. The time spent exploring and sniffing each object was recorded. The NOR index was determined as the time spent interacting with the novel object divided by the total time of exploration during the testing phase. Animals whose exploration time was considered insufficient to allow recognition (<10 seconds per object) during the familiarization phase were excluded from analysis.
The dark-light box emergence test was used to evaluate the anxiety-like behavior and was performed as previously described
[20 (link)]. Mice were initially placed in the center of the dark chamber and had free access to the illuminated chamber. The total time spent in the illuminated chamber and the number of alternation between sides were recorded for 5 minutes. A reduction in the number of alternations or in the time spent in the illuminated compartment was interpreted as increased anxiety.
Mice were tested for spatial memory using a Barnes maze (San Diego Instruments, San Diego, CA, USA)
[21 (link)]. The 3xTg-AD female mice were tested individually over a 5-day period. Each animal was placed in the center of the maze and subjected to aversive stimuli (bright light and noise). The mouse was given the opportunity to leave the maze through the escape hole. On training days 1 to 4, mice were subjected to 4 × 3-minute trials per day (inter-trial interval time of 20 minutes). For probe trial on Day 5, mice were tested during a 90-second period. Animals were evaluated for their ability to remember the fixed position of an escape compartment. The latency and number of errors before reaching the target hole were recorded for the training and probe phases. For more consistency between animals, all training sessions took place between 7:00 and 12:00 a.m. The mice were subjected to the Barnes maze at the end of the treatment period and the animals were injected with IVIg on Day 2 and 4 of the training session, late in the afternoon to reduce the stress, and sacrificed on Day 5, after the probe trial.
The open field testing measured the general locomotor activity. The open field apparatus consists of ten Plexiglas cages with white translucent walls (80 cm × 80 cm). Movements were tracked by the automated recording of photobeam breaks (San Diego Instruments) to measure horizontal (for example, distance traveled) and vertical activity (for example, rearing). Mice were placed individually in the center of the open field and movements were recorded for 1 hour.

All studies were approved by the IACUC of Texas Tech University Health Sciences Center, Lubbock, Texas (IACUC protocol# 20026). Experiments were performed in accordance with relevant guidelines and regulations. Female CD1 pregnant mice (Charles River Laboratories, Inc., Wilmington, MA; Cat# CRL: 22, RRID: IMSR_CRL:22) and after delivery their offspring were kept under standardized light and dark conditions (12 h), humidity (70%), and temperature (22 °C). Pregnant mice were singly housed. Offspring were separated into male and female after weaning (postnatal day 21–22) and housed in a group of 2–5. They were given ad libitum access to food and water. Animal behavior was monitored daily to minimize animal suffering. We applied the following exclusion criteria to our experiments: severe weight loss, infections, or significant behavioral deficits (decreased mobility, seizures, lethargy). No animal was excluded from this study. The research design is depicted in Fig. 1. A total number of 176 (n = 16 for mother and n = 160 for offspring) mice were used to perform this study. All experiments were conducted in compliance with the ARRIVE guidelines.Fig. 1

Study design. Pregnant CD1 were exposed to Blu e-cigarette from gestational day 5 (E5) to postnatal day 7 (PD7). At the end of the exposure, plasma nicotine and cotinine level were measured by LCMS/MS, and body weight was measured at PD7, PD23, PD45 and PD90. Mice were sacrificed and brain was extracted at every time point to evaluate blood-brain barrier (BBB) integrity by western blot and immunofluorescence. Open field test, novel object recognition test and morris water maze test were conducted at adolescent and adult time point to evaluate hyperactivity and learning-memory function

Morris water maze (MWM)

Hippocampal spatial memory and learning memory were assessed by the Morris Water Maze (MWM) test, which was performed as previously reported [39 (link)]. Acquired data was analyzed using SMART V3.0 (Panlab Harvard Apparatus, Germany) video tracking system. 13 animals per group were utilized.

Novel object recognition test (NORT)

NORT was used to assess the hippocampal-dependent recognition memory. 13 animals per group were evaluated in a room with a circular open-field arena of 40 cm in diameter surrounded by black curtains and constant illumination (30 lx) as it has been previously detailed [40 (link)]. Data were analyzed by discrimination index (DI) which was calculated using the following equation: $$DI=\frac{Bexplorationtime-Aexplorationtime}{Totalexplorationtime}$$
All spaces were properly cleaned with 96% ethanol between animals, in order to eliminate odor or other cues. Data was measured and represented in seconds.

The behavioral tests were implemented 7–10 days after SE. The animals' memory function was determined using the novel object recognition test.¹⁹ The recognition index used was defined as: time consumed in the new object/(time consumed in the new object + time consumed in the familiar object).²⁰ Depressive‐ and anxiety‐like behaviors were evaluated using the tail suspension test (TST) and the open field test (OFT), respectively. The time spent immobile in TST, and time in the central zone in OFT were calculated using the ANY‐maze tracking system (Stoelting Co, IL).²¹