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/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, mu...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#34;-20 Mini Ball Head with Ceiling Mount for CCTV &#38; 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&#34; 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 di...

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

Research

JoVE Journal

Neuroscience

1.1K Views.  Georgia State University. 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.

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

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 ...

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 ...

Characterizing the Relationship Between Eye Movement Parameters and Cognitive Functions in Non-demented Parkinson's Disease Patients with Eye Tracking

Cognitive impairment is a common phenomenon in Parkinson&#8217;s disease that has implications on the prognosis. A simple, noninvasive and objective proxy measurement of cognitive function in Parkinson&#8217;s disease will be helpful in detecting early cognitive decline. As a physiological metric, eye movement parameter is not confounded by the subject&#39;s attributes and intelligence and can function as a proxy marker if it correlates with cognitive functions. To this end, this study explored the relationship between the eye movement parameters and performance in cognitive tests in multiple domains. In the experiment, a visual search task with eye tracking was set up, where subjects were asked to look for a number embedded in an array of alphabets scattered randomly on a computer screen. The differentiation between the number and the alphabet is an overlearned task such that the confounding effect of cognitive ability on the eye movement parameters is minimized. The average saccadic amplitude and fixation duration were captured and calculated during the visual search task. The cognitive assessment battery covered domains of frontal-executive functions, attention, verbal and visual memory. It was found that prolonged fixation duration was associated with poorer performance in verbal fluency, visual and verbal memory, allowing further exploration on the use of eye movement parameters as proxy markers for cognitive function in Parkinson&#8217;s disease patients. The experimental paradigm has been found to be highly tolerable in our group of Parkinson&#39;s disease patients and could be applied transdiagnostically to other disease entities for similar research questions.

Here, we present a protocol to study the relationship between the eye movement parameters and cognitive functions in non-demented Parkinson&#39;s disease patients. The experiment used an eye tracker to measure the saccadic amplitude and fixation duration in a visual search task. The correlation with performance in multi-domain cognitive tasks was subsequently measured.

Cognitive impairment is a common phenomenon in Parkinson&#8217;s disease that has implications on the prognosis. A simple, noninvasive and objective proxy ...

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% of the total stroke-related mortality with an important morbidity in terms of neurological outcome. A delayed cerebral vasospasm (CVS) may occur most often in association with a delayed cerebral ischemia. Different animal models of SAH are now being used including endovascular perforation and direct injection of blood into the cisterna magna or even the prechiasmatic cistern, each exhibiting distinct advantages and disadvantages. In this article, a standardized mouse model of SAH by double direct injection of determined volumes of autologous whole blood into the cisterna magna is presented. Briefly, mice were weighed and then anesthetized by isoflurane inhalation. Then, the animal was placed in a reclining position on a heated blanket maintaining a rectal temperature of 37 &#176;C and positioned in a stereotactic frame with a cervical bend of about 30&#176;. Once in place, the tip of an elongated glass micropipette filled with the homologous arterial blood taken from carotid artery of another mouse of the same age and gender (C57Bl/6J) was positioned at a right angle in contact with the atlanto-occipital membrane by means of a micromanipulator. Then 60 &#181;L of blood was injected in the cisterna magna followed by a 30&#176; downward tilt of the animal for 2 minutes. The second infusion of 30 &#181;L of blood into the cisterna magna was performed 24 h after the first one. The individual follow-up of each animal is carried out daily (careful evaluation of weight and well-being). This procedure allows a predictable and highly reproducible distribution of blood, likely accompanied by intracranial pressure elevation that can be mimicked by an equivalent injection of an artificial cerebral spinal fluid (CSF), and represents an acute to mild-model of SAH inducing low mortality.

We described in this protocol a standardized subarachnoid hemorrhage (SAH) mouse model by a double injection of autologous whole-blood into the cisterna magna. The high degree of standardization of the double-injection procedure represents a middle-to-acute model of SAH with relative safety regarding mortality.

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% ...

Dural Stimulation and Periorbital von Frey Testing in Mice As a Preclinical Model of Headache

The cranial meninges, comprised of the dura mater, arachnoid, and pia mater, are thought to primarily serve structural functions for the nervous system. For example, they protect the brain from the skull and anchor/organize the vascular and neuronal supply of the cortex. However, the meninges are also implicated in nervous system disorders such as migraine, where the pain experienced during a migraine is attributed to local sterile inflammation and subsequent activation of local nociceptive afferents. Of the layers in the meninges, the dura mater is of particular interest in the pathophysiology of migraines. It is highly vascularized, harbors local nociceptive neurons, and is home to a diverse array of resident cells such as immune cells. Subtle changes in the local meningeal microenvironment may lead to activation and sensitization of dural perivascular nociceptors, thus leading to migraine pain. Studies have sought to address how dural afferents become activated/sensitized by using either in vivo electrophysiology, imaging techniques, or behavioral models, but these commonly require very invasive surgeries. This protocol presents a method for comparatively non-invasive application of compounds on the dura mater in mice and a suitable method for measuring headache-like tactile sensitivity using periorbital von Frey testing following dural stimulation. This method maintains the integrity of the dura and skull and reduces confounding effects from invasive techniques by injecting substances through a 0.65 mm modified cannula at the junction of unfused sagittal and lambdoid sutures. This preclinical model will allow researchers to investigate a wide range of dural stimuli and their role in the pathological progression of migraine, such as nociceptor activation, immune cell activation, vascular changes, and pain behaviors, all while maintaining injury-free conditions to the skull and meninges.

The most notable symptom of migraine is severe head pain, and it is hypothesized that this is mediated by sensory neurons innervating the meninges. Here, we present a method to locally apply substances to the dura in a minimally invasive manner while using facial hypersensitivity as an output.

The cranial meninges, comprised of the dura mater, arachnoid, and pia mater, are thought to primarily serve structural functions for the nervous system.  ...

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s disease (HD) is principally driven by a CAG nucleotide repeat that encodes an expanded glutamine tract in huntingtin protein. Thus, the inhibition of polyglutamine (polyQ) aggregation and, in particular, aggregation-associated neurotoxicity is a useful strategy for the prevention of HD and other polyQ-associated conditions. This paper introduces generalized experimental protocols to assess the neuroprotective capacity of test compounds against HD using established polyQ transgenic Caenorhabditis elegans models. The AM141 strain is chosen for the polyQ aggregation assay as an age-associated phenotype of discrete fluorescent aggregates can be easily observed in its body wall at the adult stage due to muscle-specific expression of polyQ::YFP fusion proteins. In contrast, the HA759 model with strong expression of polyQ-expanded tracts in ASH neurons is used to examine neuronal death and chemoavoidance behavior. To comprehensively evaluate the neuroprotective capacity of target compounds, the above test results are ultimately presented as a radar chart with profiling of multiple phenotypes in a manner of direct comparison and direct viewing.

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Here, we present a protocol to assess the neuroprotective activities of test compounds in Caenorhabditis elegans, including polyglutamine aggregation, neuronal death, and chemoavoidance behavior, as well as an exemplary integration of multiple phenotypes.

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s ...

A Model for Epilepsy of Infectious Etiology using Theiler's Murine Encephalomyelitis Virus

One of the main causes of epilepsy is an infection of the central nervous system (CNS); approximately 8% of patients who survive such an infection develop epilepsy as a consequence, with rates being significantly higher in less economically developed countries. This work provides an overview of modeling epilepsy of infectious etiology and using it as a platform for novel antiseizure compound testing. A protocol of epilepsy induction by non-stereotactic intracerebral injection of Theiler's murine encephalomyelitis virus (TMEV) in C57BL/6 mice is presented, which replicates many of the early and chronic clinical symptoms of viral encephalitis and subsequent epilepsy in human patients. The clinical evaluation of mice during encephalitis to monitor seizure activity and detect the potential antiseizure effects of novel compounds is described. Furthermore, histopathological consequences of viral encephalitis and seizures such as hippocampal damage and neuroinflammation are shown, as well as long-term consequences such as spontaneous epileptic seizures. The TMEV model is one of the first translational, infection-driven, experimental platforms to allow for the investigation of the mechanisms of epilepsy development as a consequence of CNS infection. Thus, it also serves to identify potential therapeutic targets and compounds for patients at risk of developing epilepsy following a CNS infection.

Intracerebral infection with the Theiler's murine encephalomyelitis virus (TMEV) in C57BL/6 mice replicates many of the early and chronic clinical symptoms of viral encephalitis and subsequent epilepsy in human patients. This paper describes the virus infection, symptoms, and histopathology of the TMEV model.

One of the main causes of epilepsy is an infection of the central nervous system (CNS); approximately 8% of patients who survive such an infection develop ...

A Model for Encephalomyosynangiosis Treatment after Middle Cerebral Artery Occlusion-Induced Stroke in Mice

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The brain's ability to self-heal after ischemic stroke is limited by inadequate blood supply in the impacted area. Encephalomyosynangiosis (EMS) is a neurosurgical procedure that achieves angiogenesis in patients with moyamoya disease. It involves craniotomy with placement of a vascular temporalis muscle graft on the ischemic brain surface. EMS has never been studied in the setting of acute ischemic stroke in mice. The hypothesis driving this study is that EMS enhances cerebral angiogenesis at the cortical surface surrounding the muscle graft. The protocol shown here describes the procedure and provides initial data supporting the feasibility and efficacy of the EMS approach. In this protocol, after 60 min of transient middle cerebral artery occlusion (MCAo), mice were randomized to either MCAo or MCAo + EMS treatment. The EMS was performed 3-4 h after occlusion. The mice were sacrificed 7 or 21 days after MCAo or MCAo + EMS treatment. Temporalis graft viability was measured using nicotinamide adenine dinucleotide reduced-tetrazolium reductase assay. A mouse angiogenesis array quantified angiogenic and neuromodulating protein expression. Immunohistochemistry was used to visualize graft bonding with brain cortex and change in vessel density. The preliminary data here suggest that grafted muscle remained viable 21 days after EMS. Immunostaining showed successful graft implantation and increase in vessel density near the muscle graft, indicating increased angiogenesis. Data show that EMS increases fibroblast growth factor (FGF) and decreases osteopontin levels after stroke. Additionally, EMS after stroke did not increase mortality suggesting that protocol is safe and reliable. This novel procedure is effective and well-tolerated and has the potential to provide information of novel interventions for enhanced angiogenesis after acute ischemic stroke.

The protocol aims to provide methods for encephalomyosynangiosis-grafting of a vascular temporalis muscle flap on the pial surface of ischemic brain tissue-for the treatment of non-moyamoya acute ischemic stroke. The approach's efficacy in increasing angiogenesis is evaluated using a transient middle cerebral artery occlusion model in mice.

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The ...