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
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</p...

<ol>
	<li>Hillerer, K. M., Reber, S. O., Neumann, I. D., Slaterry, 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=Exposure+to+chronic+pregnancy+stress+reverses+peripartum-associated+adaptations:+implications+for+postpartum+anxiety+and+mood+disorders.">Exposure to chronic pregnancy stress reverses peripartum-associated adaptations: implications for postpartum anxiety and mood disorders.</a> Endocrinology. 152 (10), 3930-3940 (2011).</li><li>Hillerer, K. M., Neumann, I. D., Slaterry, D. 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N., Elmore, S., Barnard, K. E., Carr, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulation+of+the+hypothalamic-pituitary-adrenal+axis+in+postpartum+depression.">Dysregulation of the hypothalamic-pituitary-adrenal axis in postpartum depression.</a> Biological Research for Nursing. 8 (3), 210-222 (2007).</li><li>Nierop, A., Bratsikas, A., Zimmermann, R., Ehlert, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+stress-induced+cortisol+changes+during+pregnancy+associated+with+postpartum+depressive+symptoms.">Are stress-induced cortisol changes during pregnancy associated with postpartum depressive symptoms.</a> Psychosomatic Medicine. 68 (6), 931-937 (2006).</li><li>Ulrich-Lai, Y. M., Herman, J. 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C., P&#233;rez-Diaz, F., Hanoun, N., Cohen-Salmon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prepartum+chronic+ultramild+stress+increases+corticosterone+and+estradiol+levels+in+gestating+mice:+Implications+for+postpartum+depressive+disorders.">Prepartum chronic ultramild stress increases corticosterone and estradiol levels in gestating mice: Implications for postpartum depressive disorders.</a> Psychiatry Research. 137 (12), 123-130 (2005).</li><li>Zoubovsky, S. 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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...

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.

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	<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 corticostero...

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 ...

Our protocol is significant because it represents a new paradigm for evaluating the effects of chronic stress, specifically psychosocial stress, on pregnancy outcomes. In this protocol, we expose pregnant mice to various psychosocial insults of different intensities in an unpredictable fashion. Previous animal model paradigms employed physical stressors or a limited number of stressors administered repetitively, which does not reflect the experience of women during pregnancy.Our chronic psychosocial stress model overcomes these limitations. This method has wide ramifications for various research areas, including maternal mental health and adverse infant development-related research, and more broadly, for understanding the developmental origins of adult disease through adverse intrauterine events. Before starting each stressor in the CGS paradigm, bring the CGS group mice from the housing room into the room designated for CGS.Expose the mice to different stressors following an 11-day stressor regimen running from gestational day 6.5 to 17.5. To expose the mice to foreign objects, randomly distribute six marbles or six Legos of different shapes into a clean static cage with mouse bedding without the mouse nestlets. Place all mice from one home cage into the static cage with foreign objects for two hours, then return the mice to their home cage.To expose the mice to predator odor, place one centimeter thick dirty bedding from female rats into a clean static cage with no mouse bedding or nestlets. Place all mice from one home cage into the static cage with dirty rat bedding for two hours, then return the mice to their home cage. To expose the mice to a cage tilt, place all mice from one home cage into a clean static cage with mouse bedding without the mouse nestlets.Keep the cage tilted at a 30 degree angle against the wall for two hours, then return the mice to their home cage. To expose the mice to frequent bedding changes, place all mice from one home cage into a clean static cage with mouse bedding without the mouse nestlets. Every 10 minutes, gently move the mice to a different clean cage and replace the mouse bedding.After two hours, return the mice to their home cage. To expose the mice to bedding removal, place all mice from one home cage into an empty, clean static cage with no mouse bedding or nestlets for two hours, then return the mice to their home cage. To expose the mice to movement on a shaker, place all mice from one home cage into a clean static cage with mouse bedding without the mouse nestlets.Place the static cage atop a reciprocal lab shaker set to 140 strokes per minute for two hours, then return the mice to their home cage. To expose the mice to lights overnight, place all mice from one home cage into a clean static cage with mouse bedding without the mouse nestlets. Keep the lights on overnight to interfere with the dark cycle.On the following day, return the mice to their home cage. To expose a mouse to new cage mates, transfer the mouse into a clean static cage with mouse bedding containing two unfamiliar female mice. Leave the mouse in the static cage with the unfamiliar cage mates overnight.On the following day, returned the mouse to its home cage with familiar cage mates. To expose the mice to wet bedding, saturate the mouse bedding in a static cage with clean water kept at 24 degrees Celsius. Place all mice from one home cage into the static cage with wet bedding overnight.On the following day, return the mice to their home cage. After the stressor regimen is complete on gestational day 17.5, single house all the experimental mice to prepare for parturition and downstream functional assessments. Exposing pregnant female mice to CGS causes changes in chronic stress-relevant parameters, such as reduction in body weight gain during pregnancy and increased adrenal gland weights in the early postpartum period.Importantly, CGS dams exhibit postpartum abnormalities and maternal neuroendocrine function as evidenced by increased serum corticosterone levels following a novel acute insult. Furthermore, 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 while the mean number of bouts is increased, indicating numerous short episodes of nurturing behavior.Sucrose preference is also depressed in CGS dams when compared to control dams, suggesting the presence of anhedonia. 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. In the offspring, exposure to CGS in utero results in decreased weight gain during the postnatal period from postnatal day 7 to 21, although there are no differences at birth.This reduction in body weight gain is present in offspring of both sexes. When attempting this procedure, establishing a schedule for obtaining timed pregnancies and consistently implementing the entire series, variable unpredictable psychosocial stressors is critical. This technique can help answer interesting questions such as can maternal psychosocial stress cause sex-specific effects on placental function or offspring brain development and behavior?And can these effects be reversed or modified by pharmacological intervention or postnatal maternal care?

Research

JoVE Journal

Behavior

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