Name: using a murine model of psychosocial stress in pregnancy as a translationally relevant paradigm for psychiatric disorders in mothers and infants
Uploaded: 2021-06-13T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Using a Murine Model of Psychosocial Stress in Pregnancy as a Translationally Relevant Paradigm for Psychiatric Disorders in Mothers and Infants at JoVE.com

The authors wish to acknowledge support from the National Institute of General Medical Sciences T32 GM063483-14 grant and Cincinnati Children&#39;s Research Foundation. For data adapted from Zoubovsky et al., 2019, Creative Common License can be found at the following location: http://creativecommons.org/licenses/by/4.0/.

All animal experiments described were approved by the Animal Care and Use Committee at Cincinnati Children&#39;s Medical Center and were in accordance with the National Institutes of Health guidelines. Ad libitum access to standard rodent chow and water was provided at all times to mice, including during the CGS paradigm. Mice were housed on a 14 h/10 h light-dark cycle (lights on 06:00 h) unless otherwise specified (i.e., exposure to lights overnight).
1. Preparing for timed matings
<ol>
	<li>At least 2 weeks prior to setting up timed matings, house the adult female mice together in a standard mouse cage (18.4 cm x 29.2 cm x 12.7 cm), four mice per cage. Label each female mouse with a specific ID number via an ear tag. 
	NOTE: C57BL6 female mice with no prior pregnancy and between 3 to 6 months of age were used for this protocol.</li>
	<li>At least 1 week prior to setting up timed matings, individually house the adult male mice to be used for mating.</li>
</ol>
2. Setting up timed matings
<ol>
	<li>Set up the timed matings at 18:00 h. Take two female mice and place them inside a cage that holds an individually housed male mouse. Separate the timed matings the following morning by 08:00 h.</li>
</ol>
3. Checking the copulatory plug, designated as gestational day 0.5 (G0.5)
<ol>
	<li>Immediately after separating timed matings, check for the presence of a copulatory plug in the female mice. The presence of a copulatory plug will mark G0.5. Allow the mouse to hold the wire grid inside the cage and gently lift it by the tail to visualize the vaginal opening. 
	NOTE: The presence of a copulatory plug indicates sexual activity has occurred but does not guarantee a pregnancy. When attempting to calculate the number of experimental mice needed, expect 50% of mice to be plugged from timed matings and a pregnancy to plug incidence of 60%-70%.</li>
	<li>Use simple visual examination to identify the presence of a copulatory plug (an opaque whitish hardened mass within or slightly protruding from the vaginal opening). If the copulatory plug is not easily identified by simple visual examination, gently insert a blunt end probe into the vaginal opening. Identify the plugs located further back in the vagina by the resistance of probe insertion.</li>
	<li>Separate the female mice with copulatory plugs and group house in standard mouse cages, 3 to 4 mice per cage.</li>
</ol>
4. Preparing for CGS paradigm
<ol>
	<li>Randomly assign cages housing female mice with copulatory plugs into two groups on G5.5: Control and CGS group. Attempt to randomize cages to have an approximately equal number of mice per group. Transfer the mice to clean standard mouse cages and label with a &#34;do not disturb&#34; sign. Designate these cages as &#34;home cages&#34; for mice to place them at the end of each stressor.</li>
	<li>Designate a separate room in the mouse facility to perform the CGS paradigm. Design a 11-day stressor regimen, which runs from G6.5 to G17.5, to utilize each of the 7 day stressors [exposure to foreign objects (marbles or legos), predator odor exposure (dirty rat bedding), 30&#176; cage tilt, frequent changes of bedding, bedding removal, movement on shaker] twice per day, and to utilize each of the 3 night stressors (overnight lights on, cage mate change, exposure to wet bedding) overnight in a random fashion. For a possible sample schedule and schematic of experiments described below, see Figure 1. 
	NOTE: Each day stressor should fall within the light cycle of mice (lights on 06:00 h-20:00 h), and last 2 h, with at least a 2 h break in between stressors. Each night stressor should be set up at the beginning of the dark cycle (lights off 20:00 h) and separated at the start of light cycle (lights on 06:00 h).</li>
</ol>
5. Performing the CGS paradigm
<ol>
	<li>Set up specific stressors on a standard static cage with filtered top and water bottle in the room designated for the CGS paradigm. Prepare the number of static cages needed for the experiment depending on the number of mouse cages designated to undergo CGS during randomization. Before starting each stressor, transfer the mouse cages of the CGS group from the housing room to the CGS room. 
	NOTE: Perform the handling/transfer of mice from home cage to experimental cage and back in laminar flow hoods.</li>
	<li>Apply the following stressors according to the pre-designed regimen (refer to step 4.2).
	<ol>
		<li>Exposure to foreign objects (marbles or legos): Place six marbles (14 mm in diameter) or six legos (different shapes, not to exceed 4 cm in height) randomly distributed into a clean static cage with mouse bedding, without including the mouse nestlets. Place the mice together with their home cage counterparts into the static cage with foreign objects for 2 h. Return the mice to their home cage with the same counterparts at the conclusion of the stressor. 
		NOTE: Clean the foreign objects after use.</li>
		<li>Predator odor exposure (dirty rat bedding): Place 1 cm in depth of fresh dirty rat bedding from female rats into a clean static cage with no mouse bedding, without including the mouse nestlets. Place the mice together with their home cage counterparts into the static cage with dirty rat bedding for 2 h. Return the mice to their home cage with the same counterparts at the conclusion of the stressor.</li>
		<li>30&#176; cage tilt: Place the mice with their home cage counterparts into a clean static cage with mouse bedding, without including the mouse nestlets. Tilt the cage at 30&#176; against the wall for 2 h. Return the mice to their home cage with the same counterparts at the conclusion of the stressor.</li>
		<li>Frequent changes of bedding: Place the mice with their home cage counterparts into a clean static cage with mouse bedding, without including the mouse nestlets. Substitute the mouse bedding with clean mouse bedding every 10 min for 2 h. During mouse bedding changes, gently place the mice in a different clean cage to avoid direct contact with the mice. Return the mice to their home cage with the same counterparts at the conclusion of the stressor.</li>
		<li>Bedding removal: Place the mice together with their home cage counterparts into an empty clean static cage (with no mouse bedding or nestlets) for 2 h. Return the mice to their home cage with the same counterparts at the conclusion of the stressor.</li>
		<li>Movement on shaker: Place the mice with their home cage counterparts into a clean static cage with mouse bedding, without including the mouse nestlets. Place the static cage atop a reciprocal lab shaker set to 140 strokes per min for 2 h. Return the mice to their home cage with the same counterparts at the conclusion of the stressor.</li>
		<li>Overnight exposure to lights: Place the mice with their home cage counterparts into a clean static cage with mouse bedding, without including the mouse nestlets. Keep the lights on overnight (20:00 h-06:00 h) to interfere with dark cycle. Return the mice to their home cage with the same counterparts at the conclusion of the stressor.</li>
		<li>Cage mate change: Transfer the mouse into a clean static cage with mouse bedding which is being housed by a different group of two female mice (intact females not part of treatment or control group). Keep the mouse in the static cage with unfamiliar cage mates overnight. Return the mouse to its home cage with its specific home cage counterparts at the conclusion of the stressor.</li>
		<li>Exposure to wet bedding: Fill the static cage with mouse bedding with clean water kept at 24 &#176;C until bedding is saturated with water. Place the mice together with their home cage counterparts into the static cage with wet bedding overnight. Return the mice to their home cage with the same counterparts at the conclusion of the stressor.</li>
	</ol>
	</li>
	<li>During CGS paradigm, keep the control mice undisturbed in their home cages inside the housing room.</li>
	<li>Replace the used home cages with new home cages on G10.5. On G17.5, at the conclusion of the overnight stressor, single-house all the experimental mice to prepare for parturition and downstream functional assessments.</li>
</ol>
6. Monitoring the experimental mice during the CGS paradigm
<ol>
	<li>Monitor the mice every 1 h during stressor application, except during overnight stressors.</li>
	<li>Exclude the mice displaying distress signs, including wounds, lethargy, or any physical abnormality from the experiment. Contact the veterinary staff as needed.</li>
</ol>
7. Measuring the percentage body weight gain during gestation in the experimental mice (optional)
<ol>
	<li>On G6.5, weigh the mice individually before the exposure to stressors. On G17.5, at the conclusion of the overnight stressor, weigh the mice individually. Weigh the control mice at the equivalent gestational time points.</li>
	<li>Measure the percentage body weight gain during gestation by setting the weight of the first day of the CGS paradigm (G6.5) as 100%.</li>
</ol>
8. Measuring the postpartum relative adrenal gland weights in experimental mice (optional)
<ol>
	<li>On postpartum day 2 (PP2), weigh the control and the CGS dams individually. Euthanize the dams by carbon dioxide inhalation&#160;followed by cervical dislocation in a fume hood.</li>
	<li>Place the mice on a dissection plate, sterilize the abdominal area with 70% ethanol, and open the abdominal cavity using scissors to make a vertical cut. Isolate the adrenal glands located adjacent to the anterior pole of the kidneys with forceps, bilaterally. Carefully dissect the fat tissue surrounding the adrenal glands underneath a dissecting microscope.</li>
	<li>Weigh the bilateral adrenal glands individually. Calculate the relative adrenal gland weights in milligrams per gram (total weight of the right and the left adrenal glands/body weight).</li>
</ol>
9. Measuring the postpartum hypothalamic pituitary adrenal (HPA) axis activity in the experimental mice (optional)
<ol>
	<li>In preparation for HPA axis measurements, euthanize&#160;litters to 6 pups per litter on postpartum day 0 (PP0). Use carbon dioxide inhalation, followed by decapitation with surgical scissors as a secondary method of euthanasia.</li>
	<li>On postpartum day 2 (PP2), individually restrain the control and the CGS dams inside a well-ventilated 50 mL polypropylene conical tube for 20 min. Immediately after restraint stress, remove the mouse from the conical tube and restrain the mouse with the non-dominant hand by holding the loose skin over the shoulders and posterior to the ears to have the skin over the mandible taut.</li>
	<li>Puncture the submandibular vein with a lancet slightly behind the mandible but anterior to the ear canal. Collect up to 100 &#181;L of maternal blood in a serum separator tube. After sample collection, apply gentle pressure with gauze to the puncture site to stop the bleeding. Return the dams to the home cage once the bleeding stops.</li>
	<li>Centrifuge the serum separator tube at 21,130 x g for 6 min and carefully remove the serum. Store the serum at -20 &#176;C for later use. Measure the serum corticosterone concentration by an ELISA kit following the manufacturer&#39;s protocol.</li>
</ol>
10. Measuring the postpartum behavioral changes in the experimental mice (optional)
<ol>
	<li>To prepare for the behavioral analysis, cull litters to 6 pups per litter on PP0.</li>
	<li>Perform analysis of the maternal care fragmentation from PP2 to PP5. On each day, during the light cycle, expose the dams to the testing room for a 5 min habituation period before videotaping the maternal behavior for a 30 min period.
	<ol>
		<li>Assess the maternal care fragmentation by measuring the average length of an individual licking/grooming bout and the total number of bouts performed by dams19. 
		NOTE: Licking/grooming behavior is defined as a behavior where the dam is making contact with the pup&#39;s body with her tongue, or the pup is being handled by the dam with her forepaws. A bout is defined as an uninterrupted period of time where the dam is engaged in licking/grooming of her pups.</li>
	</ol>
	</li>
	<li>Perform analysis of anhedonia via sucrose preference test (SPT) from PP0 to PP6. Expose the dams to one 100 mL bottle of clean water and one 100 mL bottle of 4% sucrose solution in their home cage. Measure the amount of water and sucrose consumed (in mL) daily. Interchange the bottle placement in the home cage. Calculate the sucrose preference using the averages from the last 4 days: preference % = [(sucrose consumption / sucrose + water consumption) x 100].</li>
	<li>Perform analysis of anxiety-like behavior via elevated zero maze (EZM) on PP8. Place the dams individually on the EZM apparatus consisting of two closed quadrants and two open quadrants elevated from the floor. Allow the dams to explore the maze undisturbed for 5 min. Quantify the time spent in the open quadrant and the number of entries into the open quadrants.</li>
</ol>
11. Measuring the postnatal offspring weight changes (optional)
<ol>
	<li>To prepare for the offspring weight analysis, cull litters to 6 pups per litter on the day of birth (postnatal day 0, PN0).</li>
	<li>Record the weight of pups on PN0 and at different time points during the postnatal period (PN2, 7, 15, 21).</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+estrous+cycle+identification+tool+and+images.">Mouse estrous cycle identification tool and images.</a> PLOS One. 7 (4), 35538 (2012).</li><li>Pallares, P., Gonzalez-Bulnes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+ultrasound+imaging+for+early+diagnosis+of+pregnancy+and+determination+of+litter+size+in+the+mouse.">Use of ultrasound imaging for early diagnosis of pregnancy and determination of litter size in the mouse.</a> Laboratory Animals. 43 (1), 91-95 (2009).</li><li>Froberg-Fejko, K., Lecker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+environmental+enrichment+and+nutritional+supplementation+to+improve+breeding+success+in+rodents.">Using environmental enrichment and nutritional supplementation to improve breeding success in rodents.</a> Lab Animal (NY). 45 (1), 406-407 (2016).</li><li>Perani, C. V., Neumann, I. D., Reber, S. O., Slattery, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-fat+diet+prevents+adaptive+peripartum-associated+adrenal+gland+plasticity+and+anxiolysis.">High-fat diet prevents adaptive peripartum-associated adrenal gland plasticity and anxiolysis.</a> Scientific Reports. 5, 14821-14831 (2015).</li><li>Nugent, B. M., Bale, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+omniscient+placenta:+metabolic+and+epigenetic+regulation+of+fetal+programming.">The omniscient placenta: metabolic and epigenetic regulation of fetal programming.</a> Frontiers in Neuroendocrinology. 39, 28-37 (2015).</li></ol>

The authors have no conflicts of interest to disclose.

Exposing the pregnant mice to CGS perturbs postpartum maternal neuroendocrine function, including HPA axis response to novel stressors, and is associated with various behavioral abnormalities relevant to perinatal mood and anxiety disorders. Given that the model employs utilization of an environmental risk factor, higher phenotypic variation is expected than otherwise observed in genetic models22. Nevertheless, results obtained from application of the CGS paradigm can be consistent across research laboratories if care is taken to minimize variables that may confound results.
Critical steps in the protocol include steps related to general husbandry practices, including housing control mice separate from CGS mice, and timed-mating steps. Co-housing control and CGS mice could in itself be a stressful stimulus for the control group and therefore confound neuroendocrine or behavioral results23,24. Likewise, initiating experiments with pregnant mice shipped from the supplier is not recommended. To maximize the efficiency of timed matings, it is recommended to house adult female mice together at least 2 weeks prior to setting up timed matings to synchronize their estrus cycles. Likewise, using sexually experienced adult male mice and preventing males from mating at least 1 week prior to setting up timed matings will maximize their fertility and increase the potential for successful pregnancies. The timeline of the CGS paradigm must also be carefully followed. Applying these stressors of varying intensity too early in gestation could affect uterine decidualization and inhibit embryo implantation25. Stress exposure during different gestational time windows has also been found to carry varying sex-specific neurodevelopmental disease risk for offspring, where male offspring are significantly more vulnerable than female offspring to stressors during early gestation26,27. CGS schedule must be designed in such a way that it ensures unpredictability to prevent the development of adaptation mechanisms and acclimation often associated with repeated exposure to predictable stressors28. Lastly, litters should be culled to six pups on the day of birth to ensure comparable conditions across all dams and prevent litter size variabilities from confounding maternal hormone or behavior analysis. Likewise, different cohorts should be employed for neuroendocrine and behavioral assessments to minimize confounding effects of restraint stress and submandibular bleeds on behavior. Different cohorts should also be used for maternal care assessment and analysis of other behavioral parameters to minimize disruption of maternal interaction with the pups.
There are several limitations to the current protocol. The inability to accurately predict the number of pregnant mice prior to the start of the CGS paradigm can pose a considerable financial and animal use burden. Modifications could be made to the protocol to achieve more predictable success with timed matings, including evaluating vaginal cytology to identify mice in estrus stage, where both mating and ovulation typically occur29. Ultrasonographic examination of mice could also be incorporated into the CGS paradigm as an alternative non-invasive technique to accurately identify pregnancies from very early stages of gestation30. The use of special breeding chow, with increased fat content, has also been employed by other groups to improve mating success31. However, caution must be taken when instituting changes in diet, given that this could affect maternal stress reactivity and behavior32. In addition, the current protocol has been shown to be effective in wild-type C57BL/6 mice, but modifications of the protocol may be needed for different strains or genetic backgrounds as well as species, for they may have large variations in stress sensitivity, maternal care, and emotional regulation.
Compared to currently existing peripartum stress models, the CGS paradigm proves to be more translationally relevant given the resulting disease-relevant endophenotypes observed, including enhanced maternal stress reactivity and postpartum abnormalities in maternal care, anhedonia, and anxiety. These alterations seem to recapitulate clinical findings associated with perinatal mood and anxiety disorders. Future applications of this model include utilizing the CGS paradigm to identify sex-specific effects of maternal psychosocial stress on offspring brain development and disease susceptibility. Studying the effects of CGS on placental function should be considered, given that dysfunction in key placental functions have been shown to impact fetal brain development33. Incorporating cross-fostering experiments with the CGS paradigm would further help understand the individual contributions in-utero CGS exposure and associated maternal hormonal milieu changes versus postpartum abnormalities in nurturing behavior play in shaping offspring emotional development.

The mechanisms underlying increased susceptibility to neuropsychiatric disorders in mothers and infants following adverse maternal exposures in the peripartum period remain largely unknown. Substantial maternal physiological alterations occur during pregnancy and the transition to the postpartum period, including several neuroendocrine adaptations that are hypothesized to be critical not only for healthy offspring neurodevelopment but also for preserving maternal mental health1,2. At the level of the maternal hypothalamic pituitary adrenal (HPA) axis, adaptions in both circadian and stress-induced levels of glucocorticoid release are observed, including a more flattened rhythm of diurnal HPA axis activity and dampened HPA axis response to acute stressors3,4,5. Given that enhanced HPA axis activity is reported in a subset of women with postpartum affective dysregulation, including increased levels of circulating glucocorticoids and inhibited negative feedback6,7,8, exposure to stressors that result in increased postpartum stress reactivity and prevent maternal HPA axis adaptions are thought to increase susceptibility to neuropsychiatric disorders.
To elucidate the effects of stress on affective dysregulation in mothers and infants, several rodent models of stress in the peripartum period have been generated. A majority of these models are characterized by the application of physical stressors that result in homeostatic challenges and alterations in dam physiological status9, such as chronic restraint stress10 and swim stress during gestation11, or postpartum shock exposure12. Although these paradigms have been shown to result in the emergence of postpartum depressive-like behaviors and alterations in maternal care10,11,12, they have been limited by their inability to accurately capture the psychosocial nature of stressors commonly experienced by human mothers. This becomes particularly important when attempting to reveal the neuroendocrine consequences of chronic stress in the peripartum period, given that processing of different types of stressors is thought to be mediated by varying neural networks orchestrating HPA axis activation9.
In order to overcome this limitation, several groups have designed stress paradigms employing psychosocial insults or a combination of physical and psychosocial stressors. The maternal separation model, where dams are separated from her pups for several hours per day during the postpartum period13,14, and the chronic social stress model, where the dams are exposed to a male intruder in the presence of their litters15,16, have been able to reproduce the emergence of abnormalities in maternal care and depressive-like phenotypes associated with physical stress paradigms. The chronic ultramild stress paradigm, where pregnant female mice are exposed to a variety of psychosocial insults, including cage tilt and overnight illumination, as well as substantial physiological insults, such as restraint stress and food restriction, has further revealed exposure to a mixed nature of stressors results in abnormalities in maternal behavior, including impairments in maternal aggression, as well as dysregulation in the circadian activity of the HPA axis17,18. Consistent with these results, an alternating restraint stress and overcrowding model during gestation results in elevations in postpartum maternal circadian corticosterone levels as well as alterations in maternal care, although no differences are observed in HPA axis re-activity following postpartum exposure to novel acute insults1.
An expansion of this work, generating a gestational stress paradigm that employs multiple psychosocial insults presented in an unpredictable fashion and minimizes the use of physiological stressors. Studies have previously shown this chronic psychosocial stress paradigm (CGS) results in the development of maternal HPA axis dysfunction, including enhanced stress reactivity in the early postpartum period19. These changes are associated with abnormalities in maternal behavior, including alterations in the quality of maternal care received by pups, and the emergence of anhedonic and anxiety-like behaviors19, features consistent with perinatal mood and anxiety disorders20,21. Furthermore, offspring weight gain reduces during the postnatal period following in-utero exposure to CGS19, suggesting CGS may have persistent negative programming effects in future generations.
The goal in developing the CGS paradigm was to primarily utilize clinically relevant stressors, which accurately capture the type, intensity, and frequency of insults often associated with neuroendocrine dysregulation and the development of perinatal mood and anxiety disorders. Here, the study provides a detailed protocol of how to subject pregnant female mice to CGS, as well as downstream assessments that can be used to test the validity of the model.

<table><tbody><tr><td>Animal lancet</td><td>Braintree Scientific Inc.</td><td>GR4MM</td><td></td></tr><tr><td>Blunt end probe</td><td>Fine Science Tools</td><td>10088-15</td><td>Used to check for copulatory plugs</td></tr><tr><td>Bottles for SPT</td><td>Braintree Scientific Inc.</td><td>WTRBTL S-BL</td><td>100 mL glass water bottle with stopper and sipper ball point tube, graduted by 1 mL.</td></tr><tr><td>Conical tubes (50 mL)</td><td>Corning Inc.</td><td>352098</td><td>Used for restraining mice to measure HPA axis response to acute stress. Make sure conical tube has small opening at the end for ventilation.</td></tr><tr><td>Legos</td><td>Amazon</td><td>-</td><td></td></tr><tr><td>Marbles</td><td>Amazon</td><td>-</td><td></td></tr><tr><td>Mouse Corticosterone ELISA kit</td><td>Biovendor</td><td>RTC002R</td><td></td></tr><tr><td>Mouse EZM</td><td>TSE Systems</td><td>-</td><td></td></tr><tr><td>Reciprocal laboratory shaker</td><td>Labnet international</td><td>S2030-RC-B</td><td></td></tr><tr><td>Serum separator tubes</td><td>Becton Dickinson</td><td>365967</td><td></td></tr><tr><td>Static cage- bottom</td><td>Alternative Design Manufacturing and Supply Inc.</td><td>RC71D-PC</td><td></td></tr><tr><td>Static cage - filtered ventilated tops</td><td>Alternative Design Manufacturing and Supply Inc.</td><td>FT71H-PC</td><td></td></tr></tbody></table>

using a murine model of psychosocial stress in pregnancy as a translationally relevant paradigm for psychiatric disorders in mothers and infants

The peripartum period is considered a sensitive period where adverse maternal exposures can result in long-term negative consequences for both mother and offspring, including the development of neuropsychiatric disorders. Risk factors linked to the emergence of affective dysregulation in the maternal-infant dyad have been extensively studied. Exposure to psychosocial stress during pregnancy has consistently emerged as one of the strongest predictors. Several rodent models have been created to explore this association; however, these models rely on the use of physical stressors or a limited number of psychosocial stressors presented in a repetitive fashion, which do not accurately capture the type, intensity, and frequency of stressors experienced by women. To overcome these limitations, a chronic psychosocial stress (CGS) paradigm was generated that employs various psychosocial insults of different intensity presented in an unpredictable fashion. The manuscript describes this novel CGS paradigm where pregnant female mice, from gestational day 6.5 to 17.5, are exposed to various stressors during the day and overnight. Day stressors, two per day separated by a 2 h break, range from exposure to foreign objects or predator odor to frequent changes in bedding, removal of bedding, and cage tilting. Overnight stressors include continuous light exposure, changing cage mates, or wetting bedding. We have previously shown that exposure to CGS results in the development of maternal neuroendocrine and behavioral abnormalities, including increased stress reactivity, the emergence of fragmented maternal care patterns, anhedonia, and anxiety-related behaviors, core features of women suffering from perinatal mood and anxiety disorders. This CGS model, therefore, becomes a unique tool that can be used to elucidate molecular defects underlying maternal affective dysregulation, as well as trans-placental mechanisms that impact fetal neurodevelopment and result in negative long-term behavioral consequences in the offspring.

Exposing the pregnant female mice to CGS results in changes in chronic stress-relevant parameters, including a reduction in body weight gain during pregnancy (Figure 2A) and increased adrenal gland weights in the early postpartum period (Figure 2B)19. Importantly, exposure to CGS results in postpartum abnormalities in maternal neuroendocrine function. CGS dams exhibit a hyperactive HPA axis as evidenced by the increased serum corticosterone levels following the application of a novel acute insult (Figure 3)19.
Exposing the pregnant female mice to CGS further results in behavioral abnormalities in the early postpartum period that appear to reflect the emergence of a depressive-like phenotype. CGS dams display alterations in maternal care as reflected by an increase in the degree of fragmentation of maternal signals received by the pups. The average duration of licking/grooming bouts is reduced and associated with an increase in the mean number of bouts following CGS, indicating numerous short episodes of nurturing behavior (Figure 4A,B)19. Sucrose preference is also depressed in CGS dams when compared to control dams, suggesting the presence of anhedonia (Figure 4C)19. Lastly, the CGS dams also display increased anxiety-related behaviors as measured by a reduction in the time spent in the open quadrants of the EZM when compared to control dams (Figure 4D)19.
In the offspring, exposure to CGS in-utero results in decreased weight gain during the postnatal period, from postnatal day 7 to 21, although no changes are observed at birth. This reduction in body weight gain is present in offspring of both sexes (Figure 5)19. Of note, the CGS paradigm did not have any effect on gestational length, litter size, or sex ratio per litter (data not shown)19.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62464/62464fig01.jpg" /> 
Figure 1: Schematic of CGS paradigm and functional assessments for validation. This figure has been modified from Zoubovsky, S.P. et al.19. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62464/62464fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62464/62464fig02.jpg" /> 
Figure 2: Changes in the chronic stress-related parameters in the dams following CGS exposure. (A) Body weight changes from G6.5-G17.5, Control = 17, CGS = 17. (B) Relative maternal adrenal gland weights at PP2, Control = 20, CGS = 15. Data presented as mean + SEM. *p &#60; 0.05, ****p &#60; 0.0001. This figure has been modified from Zoubovsky, S.P. et al.19. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62464/62464fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62464/62464fig03.jpg" /> 
Figure 3: Maternal HPA axis measurements following CGS exposure. Maternal serum corticosterone levels measured after 20 min of restraint stress on PP2, Control = 8, CGS = 5. Data presented as mean + SEM. *p &#60; 0.05. This figure has been modified from Zoubovsky, S.P. et al.19. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62464/62464fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62464/62464fig04.jpg" /> 
Figure 4: Behavioral changes in the early postpartum period in dams following CGS exposure. (A) Mean duration and (B) number of licking/grooming bouts recorded from PP2-PP5, Control = 17, CGS = 17. (C) Percent sucrose preference in SPT, Control = 17, CGS = 19. (D) Total amount of time spent in open quadrant of EZM during the 5 min period, Control = 17, CGS = 19. Data presented as mean + SEM *p &#60; 0.05, **p &#60; 0.01. This figure has been modified from Zoubovsky, S.P. et al.19. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62464/62464fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62464/62464fig05.jpg" /> 
Figure 5: Changes in the offspring body weight during postnatal development following in-utero exposure to CGS. The offspring body weight measured from PN0 to PN21, Control = 17 litters, CGS = 17 litters.Data presented as mean + SEM. *p &#60; 0.05, ****p &#60; 0.0001. This figure has been modified from Zoubovsky, S.P. et al.19. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62464/62464fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Using a Murine Model of Psychosocial Stress in Pregnancy as a Translationally Relevant Paradigm for Psychiatric Disorders in Mothers and Infants at JoVE.com

Using a Murine Model of Psychosocial Stress in Pregnancy as a Translationally Relevant Paradigm for Psychiatric Disorders in Mothers and Infants

The chronic psychosocial stress (CGS) paradigm employs clinically relevant stressors during pregnancy in mice to model psychiatric disorders of mothers and infants. Here, we provide a step-by-step procedure of applying the CGS paradigm and downstream assessments to validate this model.

The peripartum period is considered a sensitive period where adverse maternal exposures can result in long-term negative consequences for both mother and ...

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3.0K Views.  Cincinnati Children’s Hospital Medical Center. The chronic psychosocial stress (CGS) paradigm employs clinically relevant stressors during pregnancy in mice to model psychiatric disorders of mothers and infants. Here, we provide a step-by-step procedure of applying the CGS paradigm and downstream assessments to validate this model.

Video: Using a Murine Model of Psychosocial Stress in Pregnancy as a Translationally Relevant Paradigm for Psychiatric Disorders in Mothers and Infants

Using Chronic Social Stress to Model Postpartum Depression in Lactating Rodents

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals and humans. However, many animal models of depression were developed in males and are not applicable or effective in studies of postpartum females. Recent studies have reported significant effects of chronic social stress during lactation, an ethologically relevant and effective stressor, on maternal behavior, growth, and behavioral neuroendocrinology. This manuscript will describe this chronic social stress paradigm using repeated exposure of a lactating dam to a novel male intruder, and the assessment of the behavioral, physiological, and neuroendocrine effects of this model. Chronic social stress (CSS) is a valuable model for studying the effects of stress on the behavior and physiology of the dam as well as her offspring and future generations. The exposure of pups to CSS can also be used as an early life stress that has long term effects on behavior, physiology, and neuroendocrinology.

This article describes the use of chronic resident intruder social stress as an ethologically relevant paradigm to model postpartum depression and anxiety in lactating rodents.

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals ...

An Unpredictable Chronic Mild Stress Protocol for Instigating Depressive Symptoms, Behavioral Changes and Negative Health Outcomes in Rodents

Chronic, unresolved stress is a major risk factor for the development of clinical depression. While many preclinical models of stress-induced depression have been reported, the unpredictable chronic mild stress (UCMS) protocol is an established translationally-relevant model for inducing behavioral symptoms commonly associated with clinical depression, such as anhedonia, altered grooming behavior, and learned helplessness in rodents. The UCMS protocol also induces physiological (e.g., hypercortisolemia, hypertension) and neurological (e.g., anhedonia, learned helplessness) changes that are clinically associated with depression. Importantly, UCMS-induced depressive symptoms can be ameliorated through chronic, but not acute, treatment with common SSRIs. As such, the UCMS protocol offers many advantages over acute stress protocols or protocols that utilize more extreme stressors. Our protocol involves randomized, daily exposures to 7 distinct stressors: damp bedding, removal of bedding, cage tilt, alteration of light/dark cycles, social stresses, shallow water bath, and predator sounds/smells. By subjecting rodents 3-4 hr daily to these mild stressors for 8 weeks, we demonstrate both significant behavioral changes and poor health outcomes to the cardiovascular system. This approach allows for in-depth interrogation of the neurological, behavioral, and physiological alterations associated with chronic stress-induced depression, as well as for testing of new potential therapeutic agents or intervention strategies.

The unpredictable chronic mild stress (UCMS) protocol is a validated method for studying behavioral and physiological changes associated with chronic stress and depressive symptoms. Eight weeks of imposition of the UCMS protocol induces behavioral changes and poor health outcomes in rodents of either gender.

Chronic, unresolved stress is a major risk factor for the development of clinical depression. While many preclinical models of stress-induced depression ...

A Chronic Immobilization Stress Protocol for Inducing Depression-Like Behavior in Mice

Depression is not yet fully understood, but various causative factors have been reported. Recently, the prevalence of depression has increased. However, therapeutic treatments for depression or research on depression is scarce. Thus, in the present paper, we propose a mouse model of depression induced by movement restriction. Chronic mild stress (CMS) is a well-known technique to induce depressive-like behavior. However, it necessitates a complex procedure consisting of a combination of various mild stresses. In contrast, chronic immobilization stress (CIS) is a readily accessible chronic stress model, modified from a restraint model that induces depressive behavior by restricting movement using a restrainer for a certain period. To evaluate the depressive-like behaviors, the sucrose preference test (SPT), the tail suspension test (TST), and the ELISA assay to measure stress marker corticosterone levels are combined in the present experiment. The described protocols illustrate the induction of CIS and evaluation of the changes in behavior and physiological factors for the validation of depression.

This article provides a simplified and standardized protocol for induction of depressive-like behavior in chronically immobilized mice by using a restrainer. In addition, behavior and physiological techniques to verify induction of depression are explained.

Depression is not yet fully understood, but various causative factors have been reported. Recently, the prevalence of depression has increased. However, ...

Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic Poly(I:C)

Maternal immune activation (MIA) model is increasingly well appreciated as a rodent model for the environmental risk factor of various psychiatric disorders. Numerous studies have demonstrated that MIA model is able to show face, construct, and predictive validity that are relevant to autism and schizophrenia. To model MIA, investigators often use viral mimic polyinosinic:polycytidylic acid (poly(I:C)) to activate the immune system in pregnant rodents. Generally, the offspring from immune activated dam exhibit behavioral abnormalities and physiological alterations that are associated with autism and schizophrenia. However, poly(I:C) injection with different dosages and at different time points could lead to different outcomes by perturbing brain development at different stages. Here we provide a detailed method of inducing MIA by intraperitoneal (i.p.) injection of 20 mg/kg poly(I:C) at mid-gestational embryonic 12.5 days (E12.5). This method has been shown to induce acute inflammatory response in the maternal-placental-fetal axis, which ultimately results in the brain perturbations and behavioral phenotypes that are associated with autism and schizophrenia.

Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic PolyI:C

Maternal immune activation (MIA) is a model for an environmental risk factor of autism and schizophrenia. The goal of this article is to provide a step-by-step procedure of how to induce MIA in the pregnant mice in order to enhance the reproducibility of this model.

Maternal immune activation (MIA) model is increasingly well appreciated as a rodent model for the environmental risk factor of various psychiatric ...

Immunology and Infection

Mouse Maternal Autonomic Changes from Pregnancy to Pup-Rearing

Analysis of Electrocardiograms and Behavior in Mice from Pregnancy to Lactation Period

Changes in the mother-offspring relationship are presumably accompanied by dynamic changes in the autonomic nervous system. Although temporal measurements of autonomic activity have been performed in human mothers and infants, the analysis of long-term changes remains unexplored. Mouse mothers can form social bonds with their pups and have a short period of pregnancy and lactation, which makes them useful for the examination of physiological changes from pregnancy to pup-rearing. Therefore, a telemetry system was used for several weeks to measure the changes in the autonomic nervous system and the behavior of mouse mothers. The current results showed that an electrocardiogram (ECG) could be stably recorded regardless of the movements of mothers and parturition. ECG analysis showed that the heart rate gradually decreased from pregnancy to lactation, and sympathetic activity sharply increased as the pups developed. Furthermore, the simultaneous recording of behavior and ECG in the home cage enabled us to understand the behavior-dependent influences on the ECG, thereby revealing the characteristics of autonomic nervous activity during each behavior. Thus, the present experimental method helps to understand how the physiological characteristics of mothers change from pregnancy through pup rearing, supporting the healthy development of pups.

A simultaneous recording of autonomic activity and detailed maternal behavior of mother mice from pregnancy to lactation was achieved using a telemetry system. This method helps to understand the dynamics of the physiological and behavioral characteristics in mothers from pregnancy to weaning.

Changes in the mother-offspring relationship are presumably accompanied by dynamic changes in the autonomic nervous system. Although temporal measurements ...

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, ...

Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the age of 50, yet it is the least understood and studied chronic pelvic pain disorder. A significant subset of patients with chronic pelvic pain report having experienced early life stress or abuse, which can markedly affect the functioning and regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Mast cell activation, which has been shown to be increased in both urine and expressed prostatic secretions of CP/CPPS patients, is partially regulated by downstream activation of the HPA axis. Neonatal maternal separation (NMS) has been used for over two decades to study the outcomes of early life stress in rodent models, including changes in the HPA axis and visceral sensitivity. Here we provide a detailed protocol for using NMS as a preclinical model of CP/CPPS in male C57BL/6 mice. We describe the methodology for performing NMS, assessing perigenital mechanical allodynia, and histological evidence of mast cell activation. We also provide evidence that early psychological stress can have long-lasting effects on the male urogenital system in mice.

We are measuring perigenital mechanical sensitivity and mast cell activation in the prostate of male C57BL/6 mice that underwent an early life stress paradigm &#8211; neonatal maternal separation, in order to induce a preclinical model of chronic prostatitis/chronic pelvic pain syndrome.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the ...

Medicine

Generating a Reproducible Model of Mid-Gestational Maternal Immune Activation using Poly(I:C) to Study Susceptibility and Resilience in Offspring

Maternal immune activation (MIA) during pregnancy is consistently linked to increased risk of neurodevelopmental and neuropsychiatric disorders in offspring. Animal models of MIA are used to test causality, investigate mechanisms, and develop diagnostics and treatments for these disorders. Despite their widespread use, many MIA models suffer from a lack of reproducibility and almost all ignore two important aspects of this risk factor: (i) many offspring are resilient to MIA, and (ii) susceptible offspring can exhibit distinct combinations of phenotypes. To increase reproducibility and model both susceptibility and resilience to MIA, the baseline immunoreactivity (BIR) of female mice before pregnancy is used to predict which pregnancies will result in either resilient offspring or offspring with defined behavioral and molecular abnormalities after exposure to MIA. Here, a detailed method of inducing MIA via intraperitoneal (i.p.) injection of the double stranded RNA (dsRNA) viral mimic poly(I:C) at 12.5 days of gestation is provided. This method induces an acute inflammatory response in the dam, which results in perturbations in brain development in mice that map onto similarly impacted domains in human psychiatric and neurodevelopmental disorders (NDDs).

Generating a Reproducible Model of Mid-Gestational Maternal Immune Activation using PolyI:C to Study Susceptibility and Resilience in Offspring

Maternal infection is a risk factor for neurodevelopmental disorders. Mouse models of maternal immune activation (MIA) may elucidate infection's impact on brain development and function. Here, general guidelines and a procedure are provided to produce reliably resilient and susceptible offspring exposed to MIA.

Maternal immune activation (MIA) during pregnancy is consistently linked to increased risk of neurodevelopmental and neuropsychiatric disorders in ...

Neuroscience

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 ...

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

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.

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.

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