The authors have no acknowledgements.

Ethics statement: All procedures were approved by the Animal Ethics Committee of the University of Sydney and conformed to National Health and Medical Research Council of Australia guidelines. Procedures were performed on C57/BL6J mice which were reared at the University of Sydney Bosch Rodent Facility. All mice were housed in a single adequately-ventilated room in 21 &#176;C ambient temperature on a 12 hr light-dark cycle with lights on at 0600 hr in individually ventilated cages with ad libitum access to dry food and water. Late-pregnancy females were randomly assigned to standard or environmentally enriched housing conditions.
1. Housing (Enrichment Levels):
<ol>
	<li>Obtain 4 late-pregnancy adult female mice. Randomly assign 2 to the standard condition and place each of them into a clean standard mouse cage (overall dimensions 391 x 199 x 160 mm) containing one red mouse igloo. For the enriched condition, place the remaining 2 mice into a single clean rat-sized cage (overall dimensions 462 x 403 x 404 mm).</li>
	<li>Into the enrichment cage, place a variety of objects designed to increase sensory and motor stimulation (e.g. running wheels, visual stimuli, scented cotton balls, Velcro strips).</li>
	<li>Every 2 to 3 days move these objects about within the cage; refresh any that have been destroyed.</li>
	<li>Upon weaning at 21 days postnatal, sex animals and place into male-female segregated housing consistent with the environmental condition they were raised. For the enriched condition ensure that there are between 3 and 10 mice per cage with 2 and 5 mice for standard. Commence behavioral testing once animals reach adulthood (12&#8211;14 weeks of age).</li>
</ol>
2. Construction of the Puzzle Box
<ol>
	<li>Obtain 6 pieces of white acrylic (or other non-porous material): one 750 x 280 mm, two 280 x 250 mm, two 750 x 250 mm, and one 150 x 280 mm (see Figure 1).</li>
	<li>Obtain one piece of black acrylic 280 x 250 mm, with a 40 x 40 mm square opening cut into one side of this piece (see Figure 1).</li>
	<li>Assemble the Puzzle Box as follows: use the 750 x 280 mm piece as the bottom of the box, use 280 x 250 mm pieces as the ends of the box, and the 750 x 250 mm pieces as the sides of the box.</li>
	<li>Measure 150 mm into the box from one end, and place the black piece of acrylic across the box so that it splits it into two compartments (one large, one small) with the opening flush with the bottom of the box.</li>
	<li>Take the 150 x 280 mm piece of white acrylic and place it atop the smaller compartment of the box making sure it covers this area completely, providing a dark &#8220;goal-box&#8221; chamber. Affix this piece of acrylic to the body of the main box by hinges, or leave free to be completely removed during behavioral testing.</li>
	<li>Take 3 pieces of acrylic (three 4 x 120 mm) and join to make a &#8220;u-shaped&#8221; channel.</li>
</ol>
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/52225/52225fig1highres.jpg" width="500" /> 
Figure 1: Schematic diagram of the Puzzle Box task. The Puzzle Box is an acrylic box consisting of an open-field area (600 x 280 mm) and a sheltered goal-box area (150 x 280 mm), measuring 750 x 280 mm in all. A 40 x 40 mm opening within the partition (grey) separating the two areas allows animals to access to the covered goal-box area from the open field. This opening is blocked with obstructions that are increasingly difficult to remove as testing progresses. Animals undergo a five-day protocol, consisting of four obstruction conditions with three trials for each condition.
3. Running of the Puzzle Box Task
<ol>
	<li>Thoroughly clean the Puzzle Box with 70% alcohol. Repeat this step between each animal tested.</li>
	<li>Place a clean red mouse igloo within the goal-box area of the Puzzle Box, and replace the lid on the goal-box.</li>
	<li>If there is an obstruction condition being tested, place the obstruction within the doorway of the goal-box (see Figure 2).</li>
	<li>Place the mouse being tested into the open-field section of the Puzzle Box, oriented towards the goal-box, and at the end furthest from the goal-box.</li>
	<li>Record the time taken for all four paws of the animal to enter the goal-box section of the Puzzle Box.</li>
	<li>If an animal does not enter the goal-box, terminate the trial once the set time limit is reached (see Table 1).</li>
	<li>Once a trial is finished, remove the animal from the Puzzle Box and place it into a separate holding cage until the next trial begins. Keep a 60 to 180 sec gap between trials for each animal.</li>
	<li>For each animal, perform three trials per day for five consecutive days of testing, with four obstruction conditions and three trials of each condition. The third trial of a given obstruction condition should&#160;always administered on a subsequent day (see Table 1).</li>
</ol>
<img alt="Table 1" fo:content-width="5in" src="/files/ftp_upload/52225/52225table1.jpg" width="500" /> 
Table 1: Scheme of the Puzzle Box task. The Puzzle Box task is run for five days, and consists of four obstruction conditions. There are three trials on each of the first four days, and one on the fifth day. Each obstruction condition has three trials; the first two on one day, and the third the day immediately following. The first trial of an obstruction condition aims to test native problem-solving ability, the second trial examines task acquisition and reinforcement, and the third trial is used as an assay for solution retention and recall.
<img alt="Figure 2" fo:content-width="6in" src="/files/ftp_upload/52225/52225fig2highres.jpg" width="600" /> 
Figure 2: Obstruction conditions within the Puzzle Box task. Schematic diagrams&#160;of the Puzzle Box arena and the obstruction conditions used within this study. (A) Condition 0 (C0), with no obstruction present within the doorway between the open-field and goal-box areas. (B) Condition 1 (C1), with a &#8220;u shaped&#8221; channel present within the doorway between the open-field and goal-box areas. (C) Condition 2 (C2), where the channel is filled with clean bedding material. (D) Condition 3 (C3), with a &#8220;tissue plug&#8221; present within the doorway between the open-field and goal-box areas. (E) Condition 4 (C4) with a &#8220;foam plug&#8221; present within the doorway between the open-field and goal-box areas. Dimensions of the arena are as per listed in Methods and Figure 1.
4. Interpretation of Data
<ol>
	<li>Represent data as either time taken to complete the trial (including null trials where animals did not complete the task within the specified time limit) or as the number of null trials.</li>
	<li>Use a repeated measures ANOVA to assess the effect of housing condition upon performance within the Puzzle Box, with obstruction type/condition (C) and task number (T) within-subjects factors and enrichment level (standard versus enriched) as between-subjects factor.</li>
</ol>

The authors declare they have no competing financial interests.

The data presented demonstrate that the Puzzle Box can be used effectively to assess the impact of EE. Mice raised in enriched environments consistently took significantly less time to solve obstruction puzzles within this behavioral assay than did animals raised within standard laboratory conditions. Moreover, this difference was most prominent in the first trial for three of the four conditions tested, suggesting EE has a greater influence on an animal&#8217;s native problem-solving ability, relative to their capacity to reinforce or retain solutions to the problems presented by the task.
The major advantages of the Puzzle Box are its inexpensive material cost, simplicity in terms of construction and implementation, as well as a lack of need for prior training of the subjects to be tested. Further, the method can be adapted to utilize a variety of obstruction materials and conditions. For example, the protocol used here was adapted from previous studies that employed the Puzzle Box to assess cognitive ability in a variety of murine disease state models 5-7. Other studies have previously utilized variations of the Puzzle Box to assess the impact of cognitive-enhancing antipsychotics 33 and observational learning within this testing arena 34. The Puzzle Box therefore offers a behavioral task capable of assessing a wide variety of environmental, genetic and pharmacological manipulations, whilst being relatively time and cost effective.
This inherent flexibility, however, highlights the need for several key steps to successfully implement the task. As the method involves the physical removal of obstacles from a specific opening within the test arena, preliminary trials to determine which obstruction conditions are suitable and solvable within defined time limits by the animals to be assessed is critically important. This is particularly relevant when applying the task to determine the potential role of enrichment on animal models of neurodegenerative disorders whose motor abilities may be severely compromised 1-4,16,17. Moreover, multiple trials across different time intervals are required to thoroughly assess the cognitive abilities of the subjects being tested. Although task acquisition and retention are related, they can be considered as separate processes 35-37. As the findings in this study reveal, significant differences in performance can be greater within one of the factors assessed.
Although latency was the main metric used to monitor animal performance in this study, since the protocol includes video recording of all mice engaged in the task, it is also possible to perform a more detailed analysis of behavior within the testing apparatus. The way subjects from enriched and standard raised groups behave within the arena, including the manner in which they approach obstacles at each phase of the task may reveal further, more subtle differences in performance between the two cohorts 38-43. Combined with the capacity to adjust obstacles to accommodate the animals being tested, the Puzzle Box has the potential to provide a rapid and straightforward means of gaining insight into the influence of environmental factors on a range of cognitive behaviors.
Although simple and powerful, the Puzzle Box is not a substitute for a more thorough analysis of cognitive function. Instead, it provides a rapid and reliable first-pass assessment of problem solving, as well as task acquisition and recall that should then be examined more thoroughly using conventional learning tasks. The importance of such a method cannot be overstated. Traditional learning tasks can require a considerable amount of familiarizing and training of subjects before they can yield interpretable results, which may itself impact upon performance 44,45. Thus, an efficient and reliable method for obtaining a preliminary assessment of cognitive function that can be easily modified to the needs of individual experiments, such as the Puzzle Box, is highly advantageous.
Given that methods to alleviate and reverse the deleterious symptoms exhibited by transgenic models of disease states are being continually developed 46,47, a rapid and reliable means of assessing the effectiveness of the interventions from a behavioral perspective is critical. The data presented here suggests that this Puzzle Box is a useful tool that will enable such assessments.

Environmental enrichment (EE) may be defined as surroundings that provide animals with increased opportunity for social interaction, motor activity, and greater sensory stimulation than usually experienced in a standard laboratory environment.EE has been shown to consistently affect the behavior of animals, bringing about changes such as reduction of stress and anxiety-related activity 8-10, improved performance in learning and memory tasks 8,11, early onset of motor coordination and exploratory activity 11, changes in maternal care 8 as well as resistance to addictive substances 12-15. Further, EE has been revealed to ameliorate the effects of neurodegenerative disorders, delaying the onset and decreasing the severity of symptoms in animal models of Huntington&#8217;s 1-4,16, Parkinson&#8217;s 17 and Alzheimer&#8217;s disease 18.
These changes correlate with the anatomical and molecular alterations EE is known to induce throughout the brain. Animals raised in enriched environments from early stages of development show a myriad of neural changes, including increased brain weight and cortical thickness, dendritic branching 9,2-22 and synaptic density 23. EE can alter both the level and timing of growth factor expression 9,24-30, which has been shown to contribute to accelerated development of sensory 25,26,28,29, mnemonic 30, as well as motor circuits 31,32.
Previous work has revealed at times contradictory findings when investigating the impact of EE, without taking into account the different types of animals and environments used within individual studies 9,24,27,30. Currently, there is no consistent and simple behavioral task that can be used to measure the effectiveness of various EE paradigms in different strains and species of animals.
The Puzzle Box task was designed as a simple test to determine an animal&#8217;s native problem solving ability 7. Animals placed in the open area are required to remove obstructing materials situated within a small opening in order to access a covered region/shelter. Each subject is given three trials with the same obstruction in order to assess three different cognitive attributes. The first trial yields a baseline indication of inherent or native problem solving ability. The second trial, run on the same day, gives some indication of the animal&#8217;s ability to improve upon and thus reinforce strategies for removing the specific obstruction. The third trial, conducted on the following day, provides insight into the ability of the subject to retain and recall the learned solution to the task.
The motivation for solving these &#8220;obstruction puzzles&#8221; by the animals can be varied, potentially evoking an innate desire to avoid open fields and seek shelter, as well as an inherent drive to explore their surroundings 6,7. The multitude of potential behavioral drivers underlying the desire to solve the Puzzle Box suggests that various areas of the brain are involved in mediating task performance. Previous work has shown that in murine models of schizophrenia, the prefrontal cortex as well as the hippocampus are involved in the acquisition of this task 5. A lesion study in rats has also revealed a large number of brain regions involved in Puzzle Box performance, including various thalamic nuclei, the hypothalamus, the cerebellum, and limbic structures. Together, these findings indicate that engaging in this problem solving task involves a host of neural structures associated with cognitive function.
The Puzzle Box has been used successfully to assess the problem solving ability of mice, as well as cognitive deficits exhibited by murine models of schizophrenia 5-7. Performance on the task has been shown to be highly consistent, and correlate well with outcomes of other cognitive behavioral tests 6. The goal of this work was thus to adapt the Puzzle Box task to become a simple and reliable means of determining the effectiveness of EE.

<table><tbody><tr><td>Black Acrylic Board</td><td></td><td></td><td>280 x 250 mm with a 40 x 40 mm &quot;door&quot; cut into one side</td></tr><tr><td>White Acrylic Board</td><td></td><td></td><td>280 x 250 mm</td></tr><tr><td>White Acrylic Board</td><td></td><td></td><td>280 x 250 mm</td></tr><tr><td>White Acrylic Board</td><td></td><td></td><td>750 x 250 mm</td></tr><tr><td>White Acrylic Board</td><td></td><td></td><td>750 x 250 mm</td></tr><tr><td>White Acrylic Board</td><td></td><td></td><td>150 x 280 mm</td></tr><tr><td>White Acrylic Board</td><td></td><td></td><td></td></tr><tr><td>Underpass</td><td></td><td></td><td>3 pieces of 40 x 120 mm plexiglass or acrylic</td></tr><tr><td colspan="4">Note: If unable to access acrylic board, plexiglass or similar non-porous material will suffice.</td></tr><tr><td>Webcam</td><td>Logitech</td><td>C210</td><td>Fix to roof with electrical tape. Alternatively, use a tripod.</td></tr><tr><td>VirtualDub v1.10.4</td><td>VirtualDub</td><td>N/A&amp;nbsp;</td><td>Software for recording behaviour videos. Input from webcam.</td></tr><tr><td>TopScan v 3.0</td><td>CleverSys Inc.</td><td>N/A&amp;nbsp;</td><td>Software for automated top-view tracking and analysis of mouse behaviour. Captured videos are analysed post-hoc.</td></tr></tbody></table>

the use of the puzzle box as a means of assessing the efficacy of environmental enrichment

Environmental enrichment can dramatically influence the development and function of neural circuits. Further, enrichment has been shown to successfully delay the onset of symptoms in models of Huntington&#8217;s disease 1-4, suggesting environmental factors can evoke a neuroprotective effect against the progressive, cellular level damage observed in neurodegenerative disorders. The ways in which an animal can be environmentally enriched, however, can vary considerably. Further, there is no straightforward manner in which the effects of environmental enrichment can be assessed: most methods require either fairly complicated behavioral paradigms and/or postmortem anatomical/physiological analyses. This protocol describes the use of a simple and inexpensive behavioral assay, the Puzzle Box 5-7 as a robust means of determining the efficacy of increased social, sensory and motor stimulation on mice compared to cohorts raised in standard laboratory conditions. This simple problem solving task takes advantage of a rodent&#8217;s innate desire to avoid open enclosures by seeking shelter. Cognitive ability is assessed by adding increasingly complex impediments to the shelter&#8217;s entrance. The time a given subject takes to successfully remove the obstructions and enter the shelter serves as the primary metric for task performance. This method could provide a reliable means of rapidly assessing the efficacy of different enrichment protocols on cognitive function, thus paving the way for systematically determining the role specific environmental factors play in delaying the onset of neurodevelopmental and neurodegenerative disease.

The results described here are a representative sample, with data taken from several cohorts consisting of different litters. All behavioral testing was conducted between 0700 and 1100 hr, with randomized testing order of animals within a cohort. Animals raised in an enriched environment (n = 14, 7 female and 7 male) took significantly less time to solve the obstruction tasks within the Puzzle Box than those raised within a standard environment (n = 15, 7 female and 8 male) (see Figure 3) (Repeated measures ANOVA with environment as between-subjects factor, F = 19.525, p &#60;0.001). This effect of EE on performance was observed within individual trials during the Puzzle Box, where enriched mice required significantly less time to solve each of the individual obstruction puzzles, and was particularly marked during the first trial of each condition (Univariate ANOVA with environment as between-subjects factor: Condition 1-Trial 1 (C1T1), F = 4.308, p = 0.048; C1T3, F = 4.317, p = 0.047; C2T1, F = 9.466, p = 0.005; C2T2, F = 5.164, p = 0.031; C2T3, F = 7.031, p = 0.013; C3T1, F = 19.979, p = 0.000; C3T2, F = 5.788, p = 0.023; C3T3, F = 4.711, p = 0.039; C4T1, F = 5.094, p = 0.032). No effect of gender (repeated measures ANOVA with gender as between-subjects factor, F = 1.827, p = 0.188), nor any significant interaction between housing environment and gender was observed (repeated measures ANOVA with environment and gender as between-subjects factors, environment*sex, F = 0.395, p = 0.535).
<img alt="Figure 3" src="/files/ftp_upload/52225/52225fig3highres.jpg" /> 
Figure 3: Environmentally enriched mice solve the Puzzle Box faster than standard housed animals. Puzzle Box task performance of adult animals raised from birth in either enriched or standard environments, as measured by the time taken for all four paws to enter the goal-box area in seconds. Animals raised in an enriched environment (red) solved the obstruction tasks within the Puzzle Box significantly faster than those raised in a standard environment (blue) (Repeated measures ANOVA with environment as between-subjects factor, F = 19.525, p &#60;0.001). Improved performance within individual obstruction puzzle tasks was observed for enriched cohorts (Univariate ANOVA with environment as between-subjects factor: C1T1 F = 4.308, p = 0.048; C1T3, F = 4.317, p = 0.047; C2T1, F = 9.466, p = 0.005; C2T2, F = 5.164, p = 0.031; C2T3, F = 7.031, p = 0.013; C3T1, F = 19.979, p = 0.000; C3T2, F = 5.788, p = 0.023; C3T3, F = 4.711, p = 0.039; C4T1, F = 5.094, p = 0.032). C0: no obstruction; C1: U-shaped channel; C2: channel filled with bedding material; C3: tissue plug ; C4: foam plug. C1T1 refers to condition 1, trial 1 etc. (see text). Error bars: Standard error of the mean (SEM), enriched n = 14 (7 female, 7 male), standard n = 15 (7 female, 8 male). *: P &#60;0.05, **: P &#60;0.01, ***: P &#60;0.001.

Watch this Scientific Journal Video about The Use of the Puzzle Box as a Means of Assessing the Efficacy of Environmental Enrichment at JoVE.com

The Use of the Puzzle Box as a Means of Assessing the Efficacy of Environmental Enrichment

Environmental enrichment provides a potential protective effect against neurodegenerative disorders. Currently, however, there is no easy way of determining the efficacy of enrichment procedures. This protocol describes a simple &#8220;Puzzle Box&#8221; method for assessing an animal&#8217;s cognitive function, in order to reveal the effectiveness of environmental enrichment.

Environmental enrichment can dramatically influence the development and function of neural circuits.  Further, enrichment has been shown to successfully ...

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11.6K Views.  University of Sydney. Environmental enrichment provides a potential protective effect against neurodegenerative disorders. Currently, however, there is no easy way of determining the efficacy of enrichment procedures. This protocol describes a simple “Puzzle Box” method for assessing an animal’s cognitive function, in order to reveal the effectiveness of environmental enrichment.

Video: The Use of the Puzzle Box as a Means of Assessing the Efficacy of Environmental Enrichment

A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental enrichment (EE) reduces tumorigenesis by activating the mouse immune system, or affects tumor bearing animal survival by stimulating the wound repair response, including improved microbiome diversity, in the tumor microenvironment. Provided here is a detailed procedure to assess the effects of environmental enrichment on the biodiversity of the microbiome in a mouse colon tumor model. Precautions regarding animal breeding and considerations for animal genotype and mouse colony integration are described, all of which ultimately affect microbial biodiversity. Heeding these precautions may allow more uniform microbiome transmission, and consequently will alleviate non-treatment dependent effects that can confound study findings. Further, in this procedure, microbiota changes are characterized using 16S rDNA sequencing of DNA isolated from stool collected from the distal colon following long-term environmental enrichment. Gut microbiota imbalance is associated with the pathogenesis of inflammatory bowel disease and colon cancer, but also of obesity and diabetes among others. Importantly, this protocol for EE and microbiome analysis can be utilized to study the role of microbiome pathogenesis across a variety of diseases where robust mouse models exist that can recapitulate human disease.

Environmental Enrichment (EE) is an animal housing environment that is used to reveal mechanisms that underlie the connections between lifestyle, stress, and disease. This protocol describes a procedure that uses a mouse model of colon tumorigenesis and EE to specifically define alterations in microbiota biodiversity that may impact animal mortality.

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental ...

Cancer Research

A Protocol for Housing Mice in an Enriched Environment

Environmental enrichment (EE) is a housing environment for mice that boosts mental and physical health compared to standard laboratory housing. Our recent studies demonstrate that environmental enrichment decreases adiposity, increases energy expenditure, resists diet induced obesity, and causes cancer remission and inhibition in mice. EE typically consists of larger living space, a variety of &#8216;toys&#8217; to interact with, running wheels, and can include a number of other novel environmental changes. All of this fosters a more complex social engagement, cognitive and physical stimulations. Importantly, the toy location and type of toy is changed regularly, which encourages the mice to adapt to a frequently changing and novel environment. Many variables can be manipulated in EE to promote health effects in mice. Thus these approaches are difficult to control and must be properly managed to successfully replicate the associated phenotypes. Therefore, the goal of this video is to demonstrate how EE is properly set up and maintained to assure a complex, challenging, and controlled environment so that other researchers can easily reproduce the protective effects of EE against obesity and cancer.

Environmental enrichment for mice requires a complex and challenging setup, as well as comprehensive husbandry and handling techniques to assure robust metabolic and anti-cancer effects in the mice. This protocol provides detailed procedures to reproduce the above mentioned effects in mice.

Environmental enrichment (EE) is a housing environment for mice that boosts mental and physical health compared to standard laboratory housing. Our recent ...

Novel Object Exploration as a Potential Assay for Higher Order Repetitive Behaviors in Mice

Restricted, repetitive behaviors (RRBs) are a core feature of autism spectrum disorder (ASD) and disrupt the lives of affected individuals. RRBs are commonly split into lower-order and higher-order components, with lower order RRBs consisting of motor stereotypies and higher order RRBs consisting of perseverative and sequencing behaviors. Higher order RRBs are challenging to model in mice. Current assays for RRBs in mice focus primarily on the lower order components, making basic biomedical research into potential treatments or interventions for higher-order RRBs difficult. Here we describe a new assay, novel object exploration. This assay uses a basic open-field arena with four novel objects placed around the perimeter. The test mouse is allowed to freely explore the arena and the order in which the mouse investigates the novel objects is recorded. From these data, patterned sequences of exploration can be identified, as can the most preferred object for each mouse. The representative data shared here and past results using the novel object exploration assay illustrate that inbred mouse strains do demonstrate different behavior in this assay and that strains with elevated lower order RRBs also show elevated patterned behavior. As such, the novel object exploration assay appears to possess good face validity for higher order RRBs in humans and may be a valuable assay for future studies investigating novel therapeutics for ASD.

Higher order restricted, repetitive behaviors (RRBs) disrupt the lives of affected individuals. These behaviors are challenging to model in rodents, making basic biomedical research into potential treatments or interventions for RRBs difficult. Here we describe novel object exploration as a potential assay for higher order RRBs in mice.

Restricted, repetitive behaviors (RRBs) are a core feature of autism spectrum disorder (ASD) and disrupt the lives of affected individuals.  RRBs are ...

Analyzing Spatial Learning and Prosocial Behavior in Mice Using the Barnes Maze and Damsel-in-Distress Paradigms

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. The Barnes maze can also assess other mouse behaviors, such as general motivation to escape from the maze platform and exploratory behavior. The Barnes maze can measure whether a genetic mutation or environmental variable can impact the acquisition and retention of spatial memories, as well as provide information about the search strategy employed by the mice. Here we use the Barnes maze to detect a memory deficit in adult mice following a single developmental ethanol exposure event. The newly described Damsel-in-Distress paradigm exposes a male mouse to a female mouse trapped in a chamber in the open center field of the arena. It provides an opportunity for the mouse to socially respond to the trapped female and exhibit prosocial behavior. The Damsel-in-Distress paradigm can also be used to examine mouse behavior in a novel arena and measure locomotor activity. Both the Barnes Maze and the Damsel-in-Distress protocols require minimal financial investment and most aspects of the tests can be constructed from common lab supplies. These flexible and accessible tools can also be used to detect behavioral changes over the course of development.

This protocol measures spatial learning and memory using the Barnes maze. A novel Damsel-in-Distress paradigm is used to assess locomotor activity and prosocial behavior in mice.

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. ...

Assessment of Memory Function in Pilocarpine-induced Epileptic Mice

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal model of epilepsy, we generated pilocarpine-treated chronic epileptic mice. We present a protocol for three different behavioral tests using these epileptic mice: novel object location (NL), novel object recognition (NO), and pattern separation (PS) tests to evaluate learning and memory for places, objects, and contexts, respectively. We explain how to set the behavioral apparatus and provide experimental procedures for the NL, NO, and PS tests following an open field test that measures the animals&#8217; basal locomotor activities. We also describe the technical advantages of the NL, NO, and PS tests with respect to other behavioral tests for assessing memory function in epileptic mice. Finally, we discuss possible causes and solutions for epileptic mice failing to make 30 s of good contact with the objects during the familiarization sessions, which is a critical step for successful memory tests. Thus, this protocol provides detailed information about how to assess epilepsy-associated memory impairments using mice. The NL, NO, and PS tests are simple, efficient assays that are appropriate for the evaluation of different kinds of memory in epileptic mice.

This article presents experimental procedures for assessing memory impairments in pilocarpine-induced epileptic mice. This protocol can be used to study the pathophysiologic mechanisms of epilepsy-associated cognitive decline, which is one of the most common comorbidities in epilepsy.

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal ...

Simultaneous Monitoring of Wireless Electrophysiology and Memory Behavioral Test as a Tool to Study Hippocampal Neurogenesis

Brainwaves amplitude obtained from electroencephalography (EEG) has been well-recognized as a basis for cognitive capacity, memory, and learning on animals and humans. Adult neurogenesis mechanism is also linked to memory and learning improvement. Traditionally, researchers used to assess learning and memory parameters in rodent models by behavioral tasks. Therefore, the simultaneous monitoring of behavioral changes and EEG is particularly interesting in correlating data between brain activity and task-related behaviors. However, most of the equipment required to perform both studies are either complex, expensive, or uses a wired setup network that hinders the natural animals&#8217; movement. In this study, EEG was recorded with a wireless electrophysiology device along with the execution of a novel object recognition task (NORT). The animal&#8217;s behavior was monitored simultaneously by a video tracking system. Both recordings were analyzed offline by their timestamps which were synched to link EEG signals with the animal&#8217;s actions. Subjects consist of adult Wistar rats after medium-term environmental enrichment treatment. Six skull screw electrodes were fixed in pairs on both hemispheres over frontal, central, and parietal regions and were referenced to an electrode located posterior of the nasal bone. NORT protocol consists of exposing the animal to two identical objects for 10 min. After 2 h and 24 h, one of the objects was replaced with a novel one. Exploration time for each object was monitored by a behavioral tracking software (BTS) and EEG data recording. The analysis of the EEG synced with behavioral data consists of estimations of alpha and beta relative band power and comparisons between novel object recognition versus familiar object exploration, between three experimental stages. In this manuscript, we have discussed electrodes manufacturing process, epidural electrodes implantation surgery, environmental enrichment protocol, NORT protocol, BTS setup, EEG &#8211; BTS coupling for simultaneous monitoring in real-time, and EEG data analysis based on automatic events detection.

The protocol presented here provides information on the simultaneous electroencephalography (EEG) and behavioral assessment in real-time. We have discussed all steps involved in this protocol as an attractive solution for researchers in many fields of neuroscience, particularly in learning and memory areas.

Brainwaves amplitude obtained from electroencephalography (EEG) has been well-recognized as a basis for cognitive capacity, memory, and learning on ...

Using Enclosed Y-Mazes to Assess Chemosensory Behavior in Reptiles

Reptiles utilize a variety of environmental cues to inform and drive animal behavior such as chemical scent trails produced by food or conspecifics. Decrypting the scent-trailing behavior of vertebrates, particularly invasive species, enables the discovery of cues that induce exploratory behavior and can aid in the development of valuable basic and applied biological tools. However, pinpointing behaviors dominantly driven by chemical cues versus other competing environmental cues can be challenging. Y-mazes are common tools used in animal behavior research that allow quantification of vertebrate chemosensory behavior across a range of taxa. By reducing external stimuli, Y-mazes remove confounding factors and present focal animals with a binary choice. In our Y-maze studies, a scenting animal is restricted to one arm of the maze to leave a scent trail and is removed once scent-laying parameters have been met. Then, depending on the trial type, either the focal animal is allowed into the maze, or a competing scent trail is created. The result is a record of the focal animal&#39;s choice and behavior while discriminating between the chemical cues presented. Here, two Y-maze apparatuses tailored to different invasive reptile species: Argentine black and white tegu lizards (Salvator merianae) and Burmese pythons (Python bivittatus) are described, outlining the operation and cleaning of these Y-mazes. Further, the variety of data produced, experimental drawbacks and solutions, and suggested data analysis frameworks have been summarized.

Y-mazes enable researchers to determine the relevance of specific stimuli that drive animal behavior, especially isolated chemical cues from a variety of sources. Careful design and planning can yield robust data (e.g., discrimination, degree of exploration, numerous behaviors). This experimental apparatus can provide powerful insight into behavioral and ecological questions.

Reptiles utilize a variety of environmental cues to inform and drive animal behavior such as chemical scent trails produced by food or conspecifics. ...

The Innovation Arena: A Method for Comparing Innovative Problem-Solving Across Groups

Problem-solving tasks are commonly used to investigate technical, innovative behavior but a comparison of this ability across a broad range of species is a challenging undertaking. Specific predispositions, such as the morphological toolkit of a species or exploration techniques, can substantially influence performance in such tasks, which makes direct comparisons difficult. The method presented here was developed to be more robust with regard to such species-specific differences: the Innovation Arena presents 20 different problem-solving tasks. All tasks are presented simultaneously. Subjects are confronted with the apparatus repeatedly, which allows a measurement of the emergence of innovations over time - an important next step for investigating how animals can adapt to changing environmental conditions through innovative behavior.
Each individual was tested with the apparatus until it ceased to discover solutions. After testing was concluded, we analyzed the video recordings and coded successful retrieval of rewards and multiple apparatus-directed behaviors. The latter were analyzed using a Principal Component Analysis and the resulting components were then included in a Generalized Linear Mixed Model together with session number and the group comparison of interest to predict the probability of success.
We used this approach in a first study to target the question of whether long-term captivity influences the problem-solving ability of a parrot species known for its innovative behavior: the Goffin&#180;s cockatoo. We found an effect in degree of motivation but no difference in the problem-solving ability between short- and long-term captive groups.

The Innovation Arena is a novel comparative method for studying technical innovation rate per time unit in animals. It is composed of 20 different problem-solving tasks, which are presented simultaneously. Innovations can be carried out freely and the setup is robust with regard to predispositions on an individual, population or species level.

Problem-solving tasks are commonly used to investigate technical, innovative behavior but a comparison of this ability across a broad range of species is ...

Assessment of Mouse Judgment Bias through an Olfactory Digging Task

Judgment biases (JB) are differences in the way that individuals in positive and negative affective/emotional states interpret ambiguous information. This phenomenon has long been observed in humans, with individuals in positive states responding to ambiguity 'optimistically' and those in negative states instead showing 'pessimism'. Researchers aiming to assess animal affect have taken advantage of these differential responses, developing tasks to assess judgment bias as an indicator of affective state. These tasks are becoming increasingly popular across diverse species and fields of research. However, for laboratory mice, the most widely used vertebrates in research and a species heavily relied upon to model affective disorders, only one JB task has been successfully validated as sensitive to changes in affective state. Here, we provide a detailed description of this novel murine JB task, and evidence of its sensitivity to mouse affect. Though refinements are still necessary, assessment of mouse JB opens the door for answering both practical questions regarding mouse welfare, and fundamental questions about the impact of affective state in translational research.

This article provides a detailed description of a novel mouse judgment bias protocol. Evidence of this olfactory digging task's sensitivity to affective state is also demonstrated and its utility across diverse research fields is discussed.

Judgment biases (JB) are differences in the way that individuals in positive and negative affective/emotional states interpret ambiguous information. This ...

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in &#8216;enriched environments&#8217; (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of &#8216;DA&#8217; genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson&#8217;s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...