This work was supported by NIMH K99/R00 Pathway to Independence Award #MH120327, Whitehall Foundation Grant #2022-08-051, and NARSAD Young Investigator Grant #31308 from the Brain &#38; Behavior Research Foundation and The John and Polly Sparks Foundation. The authors would like to thank the Division of Animal Resources at Georgia State University for providing exceptional care to our animals., and Ryan Sleeth for his excellent technical support in setting up and maintaining our video management system. Dr. Bolton would also like to thank Dr. Tallie Z. Baram for excellent training in the proper implementation of the LBN model during her postdoctoral fellowship.

All of the procedures involving animals were performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and approved by the Institutional Animal Care and Use Committee from Georgia State University&#160;(approval number A24011). The mice were bred and maintained in the Animal Facilities at Georgia State University. The experiments were performed on a C57BL/6J strain during the perinatal period (postnatal day [P] 2-10) and included males and females. The reagents and equipment used for this study are listed in the Table of Materials.
1. Material setup
<ol>
	<li>Cut the mesh divider according to the dimensions of the cage, leaving an excess of 3 cm on the longer sides. 
	NOTE:&#160;The mesh provided in the table of materials is cut by the manufacturer.</li>
	<li>Fold the edges, including the excess, in a way that&#160;creates a platform fitted precisely to the edges of the cage and with ~2.5 cm height above the floor. This will allow urine and feces to go through the mesh divider without the animals being able to retrieve the corn cob bedding. Finally, ensure that all of the sharp edges are folded down to prevent any harm to the animals. 
	NOTE: The mesh dividers are reusable and should be thoroughly cleaned between uses with hot water and soap, followed by spraying with 70% ethanol.</li>
	<li>Set up the cameras on tripods and prepare the recording system. 
	NOTE: It is recommended to use video management software. These recordings result in .mp4 files that are automatically binned into 1 h segments.</li>
	<li>Adjust the settings to continuous recording. 
	NOTE: Additional recommended settings are 1920 x 1080 resolution and 30 frames/s.</li>
	<li>Set the resulting .mp4 files to divide into 1 h segments if not automatically done by video management software.</li>
	<li>If analyzing the lights-off period, use infrared lighting. 
	NOTE: To reduce recording glare on the side of the cage, it is recommended to disable the infrared (IR) from the camera. Instead, use IR floodlights in the room during dark phase recording.</li>
</ol>
2. Limited bedding and nesting (LBN) paradigm
<ol>
	<li>Pair 1-4 females with a single-housed male breeder. 
	NOTE: The recommended age for the first pairing in experimental females is P75. Using nulliparous females is ideal, but in situations where this is not feasible, such as with valuable transgenic mouse lines, multiparous females may continue breeding for approximately four months afterward. It is recommended, although not required, to check for vaginal plugs daily to confirm the day of mating (embryonic day [E]0).
	<ol>
		<li>Once the dams are pregnant, keep the disturbances minimal. 
		NOTE: Multiparous females can generally be reused to produce up to five litters before fertility declines. If the dams are reused for the experiment, they should preferentially be used for the same condition each time or only switched from Control to LBN, but never the other way around, in order to avoid any residual long-term effects of LBN on the dam.</li>
	</ol>
	</li>
	<li>On E17 (or if plugs were not checked, whenever the females look evidently pregnant), separate females into their own standard plexiglass cage and give them one cotton nestlet (5 x 5 cm) to build a nest. 
	NOTE: It is optimal to move separated females to a quiet, small room away from the main colony room with a 12 h light/dark cycle and minimal disruptions from personnel for the Control and LBN setup.</li>
	<li>Record the date of birth (around E19).</li>
	<li>At P2, count and sex the pups. Randomly assign litters to LBN or Control conditions (if it is the dam&#39;s first litter). This interaction with the pups should be completed between 1 and 4 h after the lights turn on.
	<ol>
		<li>Place pups carefully into a new, clean cage to be sorted by sex. The anogenital distance between the anus and genitals is greater in males than in females and can be used as an identifier.</li>
		<li>Separate males and females into groups to count them.</li>
		<li>Cull litters with more than 8 pups and discard litters with less than 4. The optimal litter size is 4-8 pups, as anything outside of this range can interfere with the distribution of maternal care during the study. 
		NOTE: The variables that can be affected due to litter size are pup weight, feeding opportunity, and maternal-pup interactions, which could confound the experiment. Ideally, mice will not be cross-fostered since they are less likely to survive than rats.</li>
	</ol>
	</li>
	<li>Set up the Control and LBN conditions as shown in Figure 1.
	<ol>
		<li>Control: Use a standard-size mouse shoebox cage (19.4 cm x 13.0 cm x 38.1 cm) with ~220 g of corn cob bedding (depending on the cage dimensions, aiming for ~1 cm height) and one standard square cotton nestlet (5 x 5 cm).
		<ol>
			<li>To help the mice acclimate to this unfamiliar environment, place one of their fecal pellets from their old cage in each corner of the new cage. In addition, place a small (dime-sized) amount of used cotton nestlet from the previous cage over the new nestlet.</li>
		</ol>
		</li>
		<li>LBN: Place the previously prepared mesh divider into a standard shoebox cage with ~110 g of corn cob bedding (aiming for ~0.5 cm height). The mesh should prevent direct contact between the mice and corn cob bedding.
		<ol>
			<li>Finally, add half of a cotton nestlet square (2.5 x 5 cm). Repeat all of the steps above to help with acclimation. 
			NOTE: It is recommended to maintain shoebox cages on standard, non-ventilated racks because it has been shown that this setup is quiet and does not induce hypothermia in pups reared in LBN cages21. However, this may not be the case with the increased airflow in ventilated cages; therefore, researchers should measure noise levels and pup core temperatures in their system if they are using ventilated cages.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Add the pups to their designated cage and place them on top of the nestlet. Next, place the dam in the cage facing the pups, as this will help her notice that they are present more quickly. Ensure that enough food and water is included for at least 8 days to avoid disturbing the cage until the end of the experiment at P10.</li>
	<li>Place a camera on a tripod in front of the cage, allowing for a clear side view of the dam and her pups. Optionally, place mirrors around the cage to better capture all of the angles. Although only 1 h of video recording per day needs to be analyzed, the camera is set up to continuously record 24/7 during the experiment so that disturbances can be monitored. 
	NOTE: Mice can also be observed in person, and maternal behavior can be hand-scored, although it is preferable to video-record to prevent any disruptions due to the presence of the experimenter in the room. If ventilated racks are used, it also may be necessary to use a different procedure to arrange cameras for maternal care recording than that described here.</li>
	<li>On the morning of P10, return all of the animals to standard caging conditions (ideally identical to the control condition above) and weigh the pups.</li>
	<li>Treat all of the pups the same and wean&#160;at P21 into groups of 2-5 same-sex littermates. 
	NOTE: Aim to house same-sex littermates together whenever possible, but if necessary, house same-sex offspring from different litters in the same condition together. It is recommended that housing control and LBN offspring together be avoided (see Yang et al. for the effect of cagemate composition on behavioral phenotypes22).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66879/66879fig01.jpg" /> 
Figure 1: Example of cage setup. The cage on the left side of the image shows a standard control (CTL) cage containing a full amount of bedding and a full nestlet. The cage on the right side shows a limited bedding and nesting (LBN) setup with half the amount of bedding, half a nestlet, and a mesh divider for separating the animals from the bedding. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66879/66879fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Maternal behavior scoring
<ol>
	<li>Although continuous recordings are collected in this setup, analyze only 1 h from P3 to P6. It is recommended to analyze videos recorded no earlier than 1 h after the lights change to allow for habituation and to analyze videos from a consistent time each day. 
	NOTE: The period from P3 to P6 typically contains the largest differences in maternal care due to LBN; therefore, it is only necessary to analyze these days. The suggested observation/recording time allows for the capture of activity after transitioning from the active to inactive cycle or vice versa. However, the inactive phase is when the dam is most likely to engage in maternal care and has previously been shown to contain the largest group differences in maternal behavior3. 
	NOTE:&#160;Capture P2 recordings later than on other days because mice need at least 1 h to habituate to the cage change. Unless this is a variable of interest, discard this day from the analysis.</li>
	<li>Score all of the behaviors as presented in Table 1. Typically, behaviors are scored for the first 50 min after the data collection time has begun. 
	NOTE: This can be done by hand or electronically using the Behavioral Observation Research Interactive Software (BORIS, an open-source software), or a similar type of software. Instructions below are for hand scoring and can be adapted for whatever software is choosen.</li>
	<li>In a printed table, record the observed behavior using the abbreviation, the start time and duration of the bout, and any descriptive notes. For example, if the dam is actively nursing and then leaves the nest but pups are still attached to her, note this as AN, followed by O. The description of the pups (and how many) still attached should be kept as a note on the side in case this is needed later. 
	NOTE: Any behavior under 3 s long is not analyzed. This rule helps filter out momentary behaviors due to outside disturbances, such as environmental noise. An exception is made when there is a visible interruption to an AN bout (such as by a large movement or stretch) that is quickly resumed. In this case, the behavior noted is AN, followed by N-AN, as described in Table 1.</li>
	<li>In the event of multiple behaviors happening at once, record the most active one. An example of this is if the dam is low nursing but later begins licking and grooming, low nursing is noted to stop, and the next behavior should be marked as LG with a note that this occurred during LN.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Type of behavior</td>
			<td>Abbreviation</td>
			<td colspan="4">Description</td>
		</tr>
		<tr>
			<td colspan="2">Licking / grooming</td>
			<td>LG</td>
			<td colspan="4">The dam is engaged in licking/grooming her pups.</td>
		</tr>
		<tr>
			<td colspan="2">Active nursing</td>
			<td>AN</td>
			<td colspan="4">The dam is nursing her pups standing up, while her back is arched.</td>
		</tr>
		<tr>
			<td colspan="2">New active nursing</td>
			<td>N-AN</td>
			<td colspan="4">This behavior is used specifically when the dam interrupted nursing but quickly resumes. This is an exception to the 3s rule.</td>
		</tr>
		<tr>
			<td colspan="2">Low nursing</td>
			<td>LN</td>
			<td colspan="4">The dam is actively nursing her pups, but her back is low or almost flat. This behavior commonly follows AN after a period of time.</td>
		</tr>
		<tr>
			<td colspan="2">Side Nursing</td>
			<td>SN</td>
			<td colspan="4">The dam is lying on her side when nursing (also known as passive nursing).</td>
		</tr>
		<tr>
			<td colspan="2">Off nest</td>
			<td>O</td>
			<td colspan="4">The dam is not on the nest, and she is not eating/drinking. This can be observed in her walking around the cage or exploring.</td>
		</tr>
		<tr>
			<td colspan="2">Eating/drinking</td>
			<td>E</td>
			<td colspan="4">The dam is off the nest eating or drinking.</td>
		</tr>
		<tr>
			<td colspan="2">Self-grooming</td>
			<td>SG</td>
			<td colspan="4">The dam is grooming herself.</td>
		</tr>
		<tr>
			<td colspan="2">Carrying pups</td>
			<td>C</td>
			<td colspan="4">The dam carries the pups, usually to relocate them back to the nest.</td>
		</tr>
		<tr>
			<td colspan="2">Nest building</td>
			<td>NB</td>
			<td colspan="4">The dam is actively constructing or relocating the nest.</td>
		</tr>
		<tr>
			<td colspan="2">Move on nest</td>
			<td>M</td>
			<td colspan="4">The dam is moving on the nest. This presents with the dam interacting with the pups in a way different than LG or any type of nursing, such as sniffing, rearing, or stepping on the pups.</td>
		</tr>
	</tbody>
</table>
Table 1: Description of the maternal care behaviors.
4. Maternal behavior data analysis
<ol>
	<li>Compile the scoring results into a spreadsheet.</li>
	<li>If the videos were analyzed electronically, delete the behaviors with bouts shorter than 3 s, as described in step 3.</li>
	<li>Calculate the average bout length, average frequency, and total duration of licking and grooming for each day of observation. 
	NOTE: Descriptive statistics can be performed on any of the scored behaviors, but licking and grooming will typically present the most evident disruption in terms of fragmentation by LBN (i.e., shorter, more frequent bouts). These variables can be analyzed as an average across P3-P6, or as repeated measures by day to look for any changes over time if this is of interest.</li>
</ol>
5. Calculation of entropy
NOTE: Entropy, or unpredictability, of maternal care behaviors is calculated based on the method proposed by Vegetabile et al.23. This method is based on the assumption that maternal care behaviors act as a Markov chain, which can be used to estimate the entropy rate of a behavioral sequence. Each dam&#39;s sequence of behaviors is characterized using the empirical transition matrix &#60;pij&#62; i,j = 1&#8230;7 of conditional probabilities of moving from one behavior (i) to another behavior (j), and the entropy rate is calculated from this as previously described3,23 and as follows: 
<img alt="Equation 1" src="/files/ftp_upload/66879/66879eq01.jpg" style="margin: auto;" /> 
where&#160;pij is the conditional probability that behavior j is observed next after a dam is observed performing behavior i, &#960;i is the frequency with which behavior i is observed, and M (=7) is the total number of different behaviors. The reader is referred to Vegetabile et al.23 for a discussion of the theoretical underpinnings of the equations; here, the focus is on how to apply the method in the LBN model.
<ol>
	<li>To compute this, arrange the format required for the analysis.
	<ol>
		<li>Combine AN, LN, SN, and N-AN into a single variable named N by adding the times together, as they all involve nursing. 
		NOTE: Refer to Table 1 for the description of the maternal care behaviors.</li>
		<li>Add the behaviors that are not related to self- or pup-directed behaviors (M and O) into O.</li>
		<li>Input LG, E, SG, C, and NB separately to result in 7 total behaviors.</li>
	</ol>
	</li>
	<li>Create a spreadsheet with columns following this order: Mouse ID, Litter ID, Litter #, Treatment, Day, Time, Behavior, and Status.
	<ol>
		<li>In the mouse ID, add the mouse line/genotype of the dam.</li>
		<li>In litter ID, use the dam&#39;s identifier followed by the litter number.</li>
		<li>In litter #, indicate the number of litters the dam has had.</li>
		<li>Ensure that the treatment is LBN or Control, according to the conditions in which the animals were placed.</li>
		<li>Indicate the day as the corresponding postnatal day analyzed.</li>
		<li>Indicate the time as the time stamp in the video when the behavior started and ended (relative to the start of recording).</li>
		<li>Select the behavior from the seven described earlier (N, O, LG, E, SG, C, and NB) (Table 1).</li>
		<li>Mark the two types of statuses, START and STOP. Therefore, each behavior will appear twice; the first notation marks the start, and the second marks the stop.</li>
	</ol>
	</li>
	<li>Following this format, include the information of all the analyzed days.</li>
	<li>In the R environment, import the datasets that have been formatted as above.</li>
	<li>Install the packages available at&#160;https://github.com/bvegetabile/entropyRate. 
	NOTE: Running this code results in a folder named LBN; regardless of the condition, this folder contains the calculated entropy. Additionally, the entropy of each day can then be averaged by the subject for P3-P6 and compared between conditions.</li>
</ol>

Modeling Early-Life Adversity in Mice Using Limited Bedding and Nesting

The authors have no conflicts of interest.

This article provides a detailed protocol to apply the LBN model in mice. This model is an important tool for understanding how an ethologically and translationally relevant form of chronic stress in early life contributes to the development of neuropsychiatric disorders in the offspring13. It is also useful for studying maternal behavior and any changes in the dams&#39; brain from a molecular, neuroendocrine, or circuit-based perspective24. For these types of questions, multiparity may be a more important variable to consider. We have observed that maternal behavior entropy scores remain consistent across multiple litters within the same dam (Figure 2C), suggesting that entropy may be a somewhat stable trait for each dam. This finding may justify the use of multiparous female mice when it is not feasible to use only nulliparous females, such as in the case of costly or rare transgenic mice. Due to the possible unintended effects of multiparity on other variables (e.g., intrauterine environment), it is recommended that the experimenter statistically control for the use of multiparous females during data analysis.
It is important to note that this model is highly sensitive to environmental disturbances. Multiple factors can disrupt the animals, causing problems and suboptimal results, such as cage flooding or loud noises such as from nearby construction. Usually, these disturbances will result in Control animals becoming stressed, and therefore, there will be no differences between the Control and LBN conditions. An indicator of these problems may be similar weights in Control and LBN pups at P10 and similar entropy values in maternal care behavior. Because of this, the ideal setup is a maternal care room that is only used for this purpose, quiet and away from the colony and other laboratory personnel. Disturbances can also lead to cannibalism of pups, especially in dams younger than P75. The risk of losing pups to cannibalism is lower in dams after P75 and often after the first litter. An alternative version of this method shifts the timing of the experimental manipulation to P4-P11 in order to decrease the risk of cannibalism even further25,26; although this is not necessary in the present laboratory conditions. Another version of LBN does not employ a mesh divider but still limits the amount of bedding and nesting material for the entire pre-weaning postnatal period27. It is important to note that changing the timeline may lead to different results, such as abusive maternal behavior, which may, in turn, result in different offspring outcomes, such as anxiety-like behaviors25,28. Other common problems include losing maternal behavior data due to the camera angle and large nest size; to help with this, it is advised to put a mirror in the back of the cage and be sure to only provide a single nestlet in the control condition in order to prevent the nest from becoming too large and concealing the pups from the camera. Finally, ensuring the mesh fits properly in the cage to prevent pups from getting stuck or hurt is essential.
An advantage of the LBN model is that it is easily customized and combined with other experimental factors. Examples of the variables that can be added to this protocol are diet29,30, immune challenges30,31, different transgenic lines32,33, and chemogenetics19. Moreover, some versions of this model employ a P10-P17 time frame and combine it with maternal separation (MS)34,35,36. This paradigm has also been adapted as a prenatal stressor, where dams are housed in the LBN environment from E14 to E1937,38, but the pups are never exposed to the stress directly. Finally, some studies use a two-hit model that involves ELA combined with different stress tests on the offspring when they reach adulthood19,39.
One of the limitations of this method is that it does not have a high throughput. The amount of data it produces is limited by the number of cameras and space available at one time, although the litters can easily be staggered over time. The manual scoring of maternal behavior is especially time-consuming and may introduce observer bias, but automated scoring is currently being developed40, thanks to the advent of machine learning-based approaches. If data are hand-scored, it is recommended that the same person scores all of the videos for one experiment in order to decrease inter-observer variability. Another limitation is that this model is designed for rodents that rely primarily on maternal care, making it difficult to study biparental care. However, some groups have adopted paternal deprivation as an alternative to MS in biparental species such as the California mouse41,42, so it is conceivable that LBN could also be adapted for these species.
Aside from LBN, maternal separation is the other most prominent model for ELA, because other forms of stress employed in adults (i.e., restraint stress) are not ethologically relevant and do not affect pups the same way. LBN differs from MS because it is a form of chronic stress that induces fragmented and unpredictable maternal behavior, whereas, in MS, deprivation is intermittent and usually occurs at predictable times each day43. Interestingly, both models can cause changes in the HPA axis and depressive-like behaviors in rodents, although LBN may be a more reproducible protocol with less experimenter interaction and higher consistency across laboratories1,11,13,16. Although this protocol is useful in mitigating some sources of variability (such as experimenter-related), other aspects like differences in animal facilities, origin of the animals, or strain can influence the results. For example, it is known that BALB/c mice have a more pronounced response to chronic restraint stress and may be differentially sensitive to ELA than C57BL/6J27,44.
In conclusion, the LBN model employs a low-resource environment with unpredictable maternal care and is a useful approach to understanding how early-life stress affects a wide variety of processes, such as physiological adaptations to stress, maternal behavior, and brain development. Notably, this model has been employed to understand how and why ELA is a risk factor for neuropsychiatric disorders1,5,9,12. In the future, the use of LBN will enhance our understanding of the biological basis of both mental and physical disorders and help elucidate novel therapeutic targets to treat the effects of stress during the sensitive perinatal period.

The early postnatal period is a critical developmental window in which environmental influences can shift the trajectory of development. For example, early-life adversity (ELA) can alter brain development to provoke long-term changes in cognitive and emotional function. Examples of ELA include physical or emotional abuse, neglect, inadequate resources, and an unpredictable home environment occurring during childhood or adolescence1. It is known that ELA is a risk factor for developing disorders such as depression, substance use disorder, post-traumatic stress disorder (PTSD), and anxiety2,3,4,5. This is important given that the levels of childhood poverty in the US have more than doubled recently, from 5.2% in 2021 to 12.4% in 20226, and although poverty itself is not necessarily ELA, it does increase the probability of various types of ELA7.
Animal models have long been essential for understanding the effects of early-life stress on brain development and adult outcomes. The two main animal models used in recent years to dissect this phenomenon are maternal separation (MS) and an impoverished environment induced by limited bedding and nesting materials (LBN). MS was developed as a model of parental deprivation8. In it, rodent dams are taken away from their pups, usually for several hours, every day until weaning8. The MS paradigm has been found to result in depressive- and anxiety-like behaviors in adulthood9, as well as an aberrant response to chronic stress10,11. On the other hand, the LBN model, first developed in the Baram laboratory12, does not separate the dam from the pups, but rather modifies the environment in which the pups are reared, mimicking a low-resource environment12,13. Decreasing the amount of nesting material and preventing direct access to the bedding in this model results in disrupted maternal care from the dams3. Since robust and predictable maternal care is required for the proper development of cognitive and emotional brain circuits14, fragmented maternal care from LBN can result in a range of outcomes, including an over-active Hypothalamic-Pituitary-Adrenal (HPA) axis, shifted excitatory-inhibitory balance in multiple brain regions, increased corticotropin-releasing hormone (CRH) levels, and depressive-like behavior in the offspring13,15,16,17,18,19.
The exact mechanism by which ELA results in increased risk for neuropsychiatric disorders is not completely understood. It is thought to be related to alterations in the HPA axis circuitry19,20, and recent evidence shows that this may be caused by changes in microglial synaptic pruning19. The LBN model has been shown to be a crucial tool for understanding the perinatal environment&#39;s impact on brain development and long-term behavioral outcomes. Although this model was initially developed for rats, it has also been adapted for mice in order to take advantage of the existing transgenic tools12,13. Notably, the model is very similar in both species and provokes highly convergent outcomes, such as alterations in the HPA axis, cognitive deficits,&#160;and depressive-like behavior, thus highlighting its cross-species utility and translational potential. This article will provide a detailed description of how to employ the limited bedding and nesting model in mice, collecting and analyzing maternal behavior and offspring outcomes to validate the model&#39;s efficacy and the expected results.

<table><tbody><tr><td>2-inch 4 MP 4x Zoom IR Mini PT Dome Network Camera</td><td>Hikvision</td><td>DS-2DE2A404IW-DE3(S6)</td><td></td></tr><tr><td>Amazon Basics Aluminum Light Photography Tripod Stand with Case - Pack of 2, 2.8 - 6.7 Feet, 3.66 Pounds, Black</td><td>Amazon</td><td></td><td>From Amazon</td></tr><tr><td>Blue Iris</td><td>Blue Iris Security</td><td></td><td>Optional video management software</td></tr><tr><td>CAMVATE 1/4&quot;-20 Mini Ball Head with Ceiling Mount for CCTV &amp;amp; Video Wall Monitors Mount - 1991</td><td>Camvate</td><td></td><td>From Amazon</td></tr><tr><td>Corn cob bedding</td><td>The Andersons</td><td>4B</td><td></td></tr><tr><td>Cotton nestlet</td><td>Ancare</td><td>NES3600</td><td></td></tr><tr><td>Mesh divider</td><td>McNICHOLS</td><td>4700313244</td><td>Standard, Aluminum, Alloy 3003-H14, 3/16&quot; No. .032 Standard (Raised), 70% Open Area</td></tr><tr><td>Tendelux DI20 IR Illuminator</td><td>Tendelux</td><td></td><td>From Amazon</td></tr></tbody></table>

limited bedding and nesting as a model for early-life adversity in mice

Early-life adversity (ELA), such as abuse, neglect, lack of resources, and an unpredictable home environment, is a known risk factor for developing neuropsychiatric disorders such as depression. Animal models for ELA have been used to study the impact of chronic stress on brain development, and typically rely on manipulating the quality and/or quantity of maternal care, as this is the major source of early-life experiences in mammals, including humans. Here, a detailed protocol for employing the Limited Bedding and Nesting (LBN) model in mice is provided. This model mimics a low-resource environment, which provokes fragmented and unpredictable patterns of maternal care during a critical developmental window (postnatal days 2-9) by limiting the amount of nesting materials given to the dam to build a nest for her pups and separating the mice from the bedding via a mesh platform in the cage. Representative data are provided to illustrate the changes in maternal behavior, as well as the diminished pup weights and long-term changes in basal corticosterone levels, that result from the LBN model. As adults, offspring reared&#160;in the LBN environment have been shown to exhibit an aberrant stress response, cognitive deficits, and anhedonia-like behavior. Therefore, this model is an important tool to define how the maturation of stress-sensitive brain circuits is altered by ELA and results in long-term behavioral changes that confer vulnerability to mental disorders.

The representative results demonstrate how ELA, imposed by an impoverished environment in LBN cages, affects maternal care from dams and offspring physiological outcomes. The daily entropy in maternal care behavior is higher in LBN across days P3-P6 (F1,58 = 7.21, p = 0.0094; Figure 2A), as well as the average entropy of each dam from this time period (t15 = 3.03, p = 0.0085; Figure 2B). Notably, there is no significant difference in average entropy rate across different litters from the same dam when maintained within the same treatment group (F1.699,4.247 = 0.57, p = 0.58; Figure 2C), suggesting that entropy rate may be a somewhat stable trait for each dam. Amongst all of the behaviors, licking and grooming is the one&#160;that has been shown to be most fragmented by LBN3. The LBN dams display a higher frequency of licking and grooming their pups (LG) (t16 = 4.04, p = 0.0010; Figure 2D) and in shorter bouts (t16 = 3.25, p = 0.0050; Figure 2E). However, there is no significant difference in the total duration of LG between the Control and LBN dams (t16 = 1.52, p = 0.15; Figure 2F).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66879/66879fig02.jpg" /> 
Figure 2: Maternal behavior analysis. (A) The daily entropy rate is higher in limited bedding and nesting (LBN) dams vs. control (CTL) as analyzed by the mixed-effects model (F1,58 = 7.21, p = 0.0094). (B) The average entropy rate is higher in LBN dams vs. CTL (t15 = 3.03, p = 0.0085). (C) The average entropy rate (P3-P6) is not significantly different across multiple litters within the same dam when maintained in the same treatment group (F1.699,4.247 = 0.57, p = 0.58). Each line represents a single dam. (D) The cumulative frequency of licking and grooming (LG) events is higher for LG dams vs. CTL (t16 = 4.04, p = 0.0010). (E) The LG average bout length is shorter for LG dams vs. CTL (t16 = 3.25, p = 0.0050). (F) The cumulative time spent on LG is not significantly different due to LBN (t16 = 1.52, p = 0.15). Data are mean &#177; SEM, * p&#160;&#60; 0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66879/66879fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Pups that were reared in LBN conditions are significantly smaller at P10 (t61 = 6.30, p &#60; 0.0001; Figure 3A). This difference typically persists at weaning age (t62 = 6.29, p = &#60;0.0001; Figure 3B) but is no longer observed by adulthood (t38 = 1.08, p = 0.29; Figure 3C). However, corticosterone levels are increased at baseline in adulthood (t18.79 = 2.23, p = 0.038; Figure 3D), suggesting enduring physiological effects of LBN. These are the expected differences in a successful experimental setup. In the case of a suboptimal setup, the values of LG bout length, entropy, and pup weights may present no differences, likely due to a stressed &#34;Control&#34; group. For the purposes of this paper, sex was collapsed when analyzing offspring outcomes because sex differences are not typically observed in the physiological outcomes described here; however, sex differences in other types of outcomes, such as cognitive and emotional behavior, are commonly reported in this model and should be investigated further4.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66879/66879fig03.jpg" /> 
Figure 3: Offspring outcomes. (A) Limited bedding and nesting (LBN) decreases pup weight measured at P10, just before return to standard cages (t61 = 6.30, p &#60; 0.0001). (B) Weight is still decreased by LBN at the weaning age (t62 = 6.29, p &#60; 0.0001). (C) Adult weight no longer differs due to LBN (t38 = 1.08, p = 0.29). (D) The baseline concentration of corticosterone in adulthood is increased by LBN, as analyzed by Welch&#39;s t-test (t18.79 = 2.23, p = 0.038). For all graphs, females are shown with circles and males with triangles. Data are mean &#177; SEM, *p &#60; 0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66879/66879fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Author Spotlight: Exploring Microglial Interactions with Stress-Response Circuitry Using the Limited Bedding and Nesting Model

This protocol describes an animal model for studying how early-life adversity, provoked by an impoverished environment and unpredictable maternal care during the early postnatal period, affects brain development and the future risk of mental disorders.

Limited Bedding and Nesting as a Model for Early-Life Adversity in Mice

Early-life adversity (ELA), such as abuse, neglect, lack of resources, and an unpredictable home environment, is a known risk factor for developing ...

limited-bedding-nesting-as-model-for-early-life-adversity

Nanyang Technological University

Research

JoVE Journal

Neuroscience

1.4K Views.  Georgia State University. In ons lab proberen we te begrijpen hoe tegenspoed in het vroege leven de ontwikkeling van de hersenen beïnvloedt en later het risico op psychische stoornissen zoals depressie en verslaving. We zijn vooral geïnteresseerd in hoe microglia interageren met stressgevoelige neuronen in meerdere hersengebieden het mechanisme kunnen zijn waardoor de herbedrading van het circuit plaatsvindt. We ontdekten eerder dat dit model voor ELA afwijkende stressreacties op vo...

Video: Author Spotlight: onderzoek naar microgliale interactie en stress-responscircuits met behulp van het Limited Bedding and Nesting-model

Nest Building as an Indicator of Health and Welfare in Laboratory Mice

The minimization and alleviation of suffering has moral and scientific implications. In order to mitigate this negative experience one must be able to identify when an animal is actually in distress. Pain, illness, or distress cannot be managed if unrecognized. Evaluation of pain or illness typically involves the measurement of physiologic and behavioral indicators which are either invasive or not suitable for large scale assessment. The observation of nesting behavior shows promise as the basis of a species appropriate cage-side assessment tool for recognizing distress in mice. Here we demonstrate the utility of nest building behavior in laboratory mice as an ethologically relevant indicator of welfare. The methods presented can be successfully used to identify thermal stressors, aggressive cages, sickness, and pain. Observation of nest building behavior in mouse colonies provides a refinement to health and well-being assessment on a day to day basis.

We demonstrate the utility of nest building behavior in laboratory mice as an indicator of welfare. Nest scoring is a sensitive technique that is altered by temperature, illness, and aggression. The time to integrate into nest test (TINT) is a simple cage-side assessment that can detect postoperative pain.

The minimization and alleviation of  suffering has moral and scientific implications. In order to mitigate this  negative experience one must be able to ...

Behavior

Nest Building Behavior as an Early Indicator of Behavioral Deficits in Mice

Nest building is an innate behavior in male and female rodents, even when raised in laboratory settings. As such, many researchers provide rodents synthetic and/or natural materials (such as twine, tissue, cotton, paper, and hay) as a gauge of their overall well-being and as an ancillary assessment to predict the possible decline in cognition. Typically, changes in nesting behaviors, such as failure to create a nest, indicate a change in health or welfare. In addition, nesting behavior is sensitive to many environmental and physiological challenges, as well as many genetic mutations underlying pathological disease states. The following protocol describes a nesting behavior paradigm that explores the usage of four types of nesting material. In addition, the protocol utilizes intraclass correlations to demonstrate that inter-rater reliability is higher when nests are constructed out of shredded paper compared to other common nesting materials such as cotton squares, paper twists, and soft cob bedding. The chosen methodology and statistical considerations (i.e., intraclass correlation) for this assay may be of interest for those conducting experiments assessing the quality of living of mice.&#160;

Here, we present a protocol to quantify nest building behavior in mice, which is known to be impaired in several neurological disorders and diseases. This protocol examines the utility of four materials and offers the opportunity to quantify the rater agreement in scoring, improving the validity and reliability of the assay.

Nest building is an innate behavior in male and female rodents, even when raised in laboratory settings. As such, many researchers provide rodents ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Modeling Early Life Trauma in Mice to Study PTSD and Social Avoidance Behavior

Chronic Social Defeat Stress in Early Adolescent Male Mice

Physical abuse and trauma in childhood is reported in as many as 1 in 7 children and is a major risk factor for the development of psychiatric diseases, such as Post-Traumatic Stress Disorder (PTSD), in adolescence and adulthood. Hallmark behavioral symptoms of PTSD include avoidance of cues and contexts associated with trauma. The ability to model long-lasting changes in the brain due to early life trauma is critical to determine potential circuit and molecular targets for the modulation of resultant symptoms. This manuscript describes a protocol for modeling early life physical and psychological trauma in early adolescent male mice that produces socially avoidant behavior. Adolescent male mice are exposed to repeated aggressive encounters followed by overnight housing, which provides an added dimension of psychological stress, with an adult male aggressor every day for 10 days. Repeated social defeat paired with overnight housing by the aggressor in early adolescence produces robust social avoidance behavior that lasts throughout adulthood. Social avoidance can be readily quantified in early adolescents, adolescents, and adults using open field social interaction testing. Early adolescent social defeat robustly produces more than 60% susceptible animals in adulthood.

This protocol details how to perform 10 day chronic social defeat stress in early adolescent male mice to produce long-lasting social avoidance in adulthood in more than 60% of mice.

Physical abuse and trauma in childhood is reported in as many as 1 in 7 children and is a major risk factor for the development of psychiatric diseases, ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded forebrain neuroepithelium is not amenable to wholemount analysis to examine organ-wide neurogenesis patterns. Moreover, defining the mechanisms of forebrain neurogenesis is not necessarily predictive of neurogenesis in other parts of the brain; for example, due to the presence of forebrain-specific progenitor subtypes. The mouse hindbrain provides an alternative model for studying embryonic neurogenesis that is amenable to wholemount analysis, as well as tissue sections to observe the spatiotemporal distribution and behavior of neural progenitors. Moreover, it is easily dissected for other downstream applications, such as cell isolation or molecular biology analysis. As the mouse hindbrain can be readily analyzed in the vast number of cell lineage reporter and mutant mouse strains that have become available, it offers a powerful model for studying the cellular and molecular mechanisms of developmental neurogenesis in a mammalian organism. Here, we present a simple and quick method to use the mouse embryo hindbrain for analyzing mammalian neural progenitor cell (NPC) behavior in wholemount preparations and tissue sections.

This article demonstrates how the mouse embryonic hindbrain can be used as a model for studying developmental neurogenesis in both whole organ and tissue section preparations.

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded ...

A Protocol for Transcranial Photobiomodulation Therapy in Mice

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide range of neurological and psychiatric disorders, and memory enhancement in age-related cognitive decline and neurodegenerative diseases. We describe a laboratory protocol for transcranial photobiomodulation therapy (PBMT) in mice. Aged BALB/c mice (18 months old) are treated with a 660 nm laser transcranially, once daily for 2 weeks. Laser transmittance data shows that approximately 1% of the incident red light on the scalp reaches a 1 mm depth from the cortical surface, penetrating the dorsal hippocampus. Treatment outcomes are assessed by two methods: a Barnes maze test, which is a hippocampus-dependent spatial learning and memory task evaluation, and measuring hippocampal ATP levels, which is used as a bioenergetics index. The results from the Barnes task show an enhancement of the spatial memory in laser-treated aged mice when compared with age-matched controls. Biochemical analysis after laser treatment indicates increased hippocampal ATP levels. We postulate that the enhancement of memory performance is potentially due to an improvement in hippocampal energy metabolism induced by the red laser treatment. The observations in mice could be extended to other animal models since this protocol could potentially be adapted to other species frequently used in translational neuroscience, such as rabbit, cat, dog, or monkey. Transcranial photobiomodulation is a safe and cost-effective modality which may be a promising therapeutic approach in age-related cognitive impairment.

Photobiomodulation therapy is an innovative noninvasive modality for the treatment of a wide range of neurological and psychiatric disorders and can also improve healthy brain function. This protocol includes a step-by-step guide to performing brain photobiomodulation in mice by transcranial light delivery, which can be adapted for use in other laboratory rodents.

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide ...

Social Isolation Model: A Noninvasive Rodent Model of Stress and Anxiety

Anxiety disorders are one of the leading causes of disability in the United States (US). Current treatments are not always effective and less than 50% of patients achieve full remission. A critical step in developing a novel anxiolytic is to develop and utilize an animal model, such as mice, to study pathological changes and test drug target(s), efficacy, and safety. Current approaches include genetic manipulation, chronic administration of anxiety-inducing molecules, or the administration of environmental stress. These methods, however, may not realistically reflect anxiety induced throughout daily life. This protocol describes a novel anxiety model, which mimics the intentional or unintentional patterns of social isolation in modern life. The social isolation-induced anxiety model minimizes perceived distractions and invasiveness and utilizes wild type C57BL/6 mice. In this protocol, 6- to 8-week-old mice (male and female) are singly housed in opaque cages to visually block the external environment, such as neighboring mice, for 4 weeks. No environmental enrichments (such as toys) are provided, bedding material is reduced by 50%, any treatment of drug is administered as an agar form, and the exposure/handling of the mice is minimized. Socially isolated mice generated using this protocol exhibit greater anxiety-like behavior, aggression,&#160;as well as decreased cognition.

Presented here is a social isolation (SI)-induced anxiety mouse model that utilizes wild type C56BL/6J mice to induce stress and anxiety-like behavior with minimal handling and no invasive procedures. This model reflects modern life patterns of social isolation and is ideal for studying anxiety and related disorders.

Anxiety disorders are one of the leading causes of disability in the United States (US). Current treatments are not always effective and less than 50% of ...

Adolescent Social Stress Paradigm in C57BL&#47;6 Mice for Behavioral Research

Social Defeat Stress Model for Adolescent C57BL&#47;6 Male and Female Mice

Social adversity in adolescence is prevalent and can negatively impact mental health trajectories. Modeling social stress in adolescent male and female rodents is needed to understand its effects on ongoing brain development and behavioral outcomes. The chronic social defeat stress paradigm (CSDS) has been widely used to model social stress in adult C57BL/6 male mice by leveraging on the aggressive behavior displayed by an adult male rodent to an intruder invading its territory. An advantage of this paradigm is that it allows to categorize defeated mice into resilient and susceptible groups based on their individual differences in social behavior 24 h after the last defeat session. Implementing this model in adolescent C57BL/6 mice has been challenging because adult or adolescent mice do not typically attack early adolescent male or female mice and because adolescence is a short period of life, encompassing discreet temporal windows of vulnerability. This limitation was overcome by adapting an accelerated version of the CSDS to be used for adolescent male and female mice. This 4-day stress paradigm with 2 physical attack sessions per day uses a C57BL/6 male adult to prime the CD-1 mouse for aggressiveness such that it readily attacks the male or female adolescent mouse. This model was termed accelerated social defeat stress (AcSD) for adolescent mice. Adolescent exposure to AcSD induces social avoidance 24 h later in both males and females, but only in a subset of defeated mice. This vulnerability occurs despite the number of attacks being consistent across sessions between resilient and susceptible groups. The AcSD model is short enough to allow exposure during discrete periods within adolescence, allows the segregation of mice according to the presence or absence of social avoidance behavior, and is the first model available to study social defeat stress in adolescent C57BL/6 female mice.

Author Spotlight: Understanding Adolescent Social Adversity Effects on Neurodevelopment in Mice

We developed an accelerated social defeat stress model for adolescent C57BL/6 mice, which works in both males and females and allows exposure during discrete adolescent periods. Exposure to this model induces social avoidance, but only in a subset of defeated male and female mice.

Social adversity in adolescence is prevalent and can negatively impact mental health trajectories. Modeling social stress in adolescent male and female ...

Limited Bedding and Nesting as a Model for Early-Life Adversity in Mice

Samenvatting

Meer video's verkennen