Mohammed R. Milad is supported by NIMH grant R01 MH097880 and institutional funds from the Department of Psychiatry at MGH.

NOTE: All procedures in this protocol have been approved by the Subcommittee on Research Animal Care, which serves as the Institutional Animal Care and Use Committee (IACUC) for the Massachusetts General Hospital, and are in compliance with the National Institutes of Health guidelines.
1. Animal Housing and Handling Procedure
<ol>
	<li>Upon arrival, house adult Sprague Dawley female rats about 8-10 weeks of age (weighing 200-225 g) in groups of 3-4 inch plastic bins with woodchip bedding. Ma...

<ol>
	<li>Baron-Cohen, S., Knickmeyer, R. C., Belmonte, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+differences+in+the+brain:+implications+for+explaining+autism.">Sex differences in the brain: implications for explaining autism.</a> Science. 310 (5749), 819-823 (2005).</li><li>Voyer, D., Voyer, S., Bryden, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnitude+of+sex+differences+in+spatial+abilities:+a+meta-analysis+and+consideration+of+critical+variables.">Magnitude of sex differences in spatial abilities: a meta-analysis and consideration of critical variables.</a> Psychological Bulletin. 117 (2), 250-270 (1995).</li><li>Pinker, S., Spelke, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Conversation+with+Steven+Pinker+and+Elizabeth+Spelke.">A Conversation with Steven Pinker and Elizabeth Spelke.</a> The Science of Gender and Science. , (2005).</li><li>Hampson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen-related+variations+in+human+spatial+and+articulatory-motor+skills.">Estrogen-related variations in human spatial and articulatory-motor skills.</a> Psychoneuroendocrinology. 15, 97-111 (1990).</li><li>Hampson, E., Kimura, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+effects+of+hormonal+fluctuations+on+human+motor+and+perceptual-spatial+skills.">Reciprocal effects of hormonal fluctuations on human motor and perceptual-spatial skills.</a> Behavioral Neuroscience. 102, 456-459 (1988).</li><li>Hampson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variations+in+sex-related+cognitive+abilities+across+the+menstrual+cycle.">Variations in sex-related cognitive abilities across the menstrual cycle.</a> Brain and Cognition. 14 (1), 26-43 (1990).</li><li>Kilpatrick, D. G., Resnick, H. S., Milanak, M. E., Miller, M. W., Keyes, K. M., Friedman, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=National+estimates+of+exposure+to+traumatic+events+and+PTSD+prevalence+using+DSM-IV+and+DSM-5+criteria.">National estimates of exposure to traumatic events and PTSD prevalence using DSM-IV and DSM-5 criteria.</a> Journal of Traumatic Stress. 26 (5), 537-547 (2013).</li><li>Breslau, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gender+differences+in+trauma+and+posttraumatic+stress+disorder.">Gender differences in trauma and posttraumatic stress disorder.</a> Journal of Gender-Specific Medicine. 5 (1), 34-40 (2002).</li><li>Frans, O., Rimmo, P. A., Aberg, L., Fredrikson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trauma+exposure+and+post-traumatic+stress+disorder+in+the+general+population.">Trauma exposure and post-traumatic stress disorder in the general population.</a> Acta Psychiatrica Scandinavica. 111 (4), 291-299 (2005).</li><li>Breslau, N., Kessler, R. C., Chilcoat, H. D., Schultz, L. R., Davis, G. C., Andreski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trauma+and+posttraumatic+stress+disorder+in+the+community:+the+1996+Detroit+Area+Survey+of+Trauma.">Trauma and posttraumatic stress disorder in the community: the 1996 Detroit Area Survey of Trauma.</a> Archives of General Psychiatry. 55 (7), 626-632 (1996).</li><li>Seedat, S., Stein, D. J., Carey, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-traumatic+stress+disorder+in+women:+epidemiological+and+treatment+issues.">Post-traumatic stress disorder in women: epidemiological and treatment issues.</a> CNS Drugs. 19 (5), 411-427 (2005).</li><li>Holbrook, T. L., Hoyt, D. B., Stein, M. B., Sieber, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gender+differences+in+long-term+posttraumatic+stress+disorder+outcomes+after+major+trauma:+women+are+at+higher+risk+of+adverse+outcomes+than+men.">Gender differences in long-term posttraumatic stress disorder outcomes after major trauma: women are at higher risk of adverse outcomes than men.</a> Journal of Trauma. 53 (5), 882-888 (2002).</li><li>Labad, J., Menchon, J. M., Alonso, P., Segalas, C., Jimenez, S., Jaurrieta, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gender+differences+in+obsessive-compulsive+symptom+dimensions.">Gender differences in obsessive-compulsive symptom dimensions.</a> Depression and Anxiety. 25 (10), 832-838 (2008).</li><li>Gupta, R. R., Sen, S., Diepenhorst, L. L., Rudick, C. N., Maren, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+modulates+sexually+dimorphic+contextual+fear+conditioning+and+hippocampal+long-term+potentiation+(LTP)+in+rats.">Estrogen modulates sexually dimorphic contextual fear conditioning and hippocampal long-term potentiation (LTP) in rats.</a> Brain Research. 888, 356-365 (2001).</li><li>Milad, M. R., Igoe, S. A., Lebron-Milad, K., Novales, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrous+cycle+phase+and+gonadal+hormones+influence+conditioned+fear+extinction.">Estrous cycle phase and gonadal hormones influence conditioned fear extinction.</a> Neuroscience. 164 (3), 887-895 (2009).</li><li>Zeidan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estradiol+modulates+medial+prefrontal+cortex+and+amygdala+activity+during+fear+extinction+in+women+and+female+rats.">Estradiol modulates medial prefrontal cortex and amygdala activity during fear extinction in women and female rats.</a> Biological Psychiatry. 70 (10), 920-927 (2011).</li><li>Lebron-Milad, K., Milad, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+differences,+gonadal+hormones+and+the+fear+extinction+network:+implications+for+anxiety+disorders.">Sex differences, gonadal hormones and the fear extinction network: implications for anxiety disorders.</a> Biology of Mood &amp; Anxiety Disorders. 2 (3), (2012).</li><li>Rey, C. D., Lipps, J., Shansky, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+d1+receptor+activation+rescues+extinction+impairments+in+low-estrogen+female+rats+and+induces+cortical+layer-specific+activation+changes+in+prefrontal-amygdala+circuits.">Dopamine d1 receptor activation rescues extinction impairments in low-estrogen female rats and induces cortical layer-specific activation changes in prefrontal-amygdala circuits.</a> Neuropsychopharmacology. 39 (5), 1282-1289 (2013).</li><li>Str&#246;m, J. O., Theodorsson, A., Ingberg, E., Isaksson, I. M., Theodorsson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovariectomy+and+17&#946;-estradiol+Replacement+in+Rats+and+Mice:+A+Visual+Demonstration.">Ovariectomy and 17&#946;-estradiol Replacement in Rats and Mice: A Visual Demonstration.</a> Journal of Visualized Experiments. , e4013 (2012).</li><li>Markham, J. A., Pych, J. C., Juraska, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+hormone+replacement+to+aged+ovariectomized+female+rats+benefits+acquisition+of+the+morris+water+maze.">Ovarian hormone replacement to aged ovariectomized female rats benefits acquisition of the morris water maze.</a> Hormones &amp; Behavior. 42 (3), 284-293 (2002).</li><li>Markowska, A. L., Savonenko, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+estrogen+replacement+in+restoration+of+cognitive+function+after+long-term+estrogen+withdrawal+in+aging+rats.">Effectiveness of estrogen replacement in restoration of cognitive function after long-term estrogen withdrawal in aging rats.</a> Journal of Neuroscience. 22 (24), 10985-10995 (2002).</li><li>Bredemann, T. M., McMahon, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=17&#946;+Estradiol+Increases+Resilience+and+Improves+Hippocampal+Synaptic+Function+in+Helpless+Ovariectomized+Rats.">17&#946; Estradiol Increases Resilience and Improves Hippocampal Synaptic Function in Helpless Ovariectomized Rats.</a> Psychoneuroendocrinology. 42, 77-88 (2014).</li><li>Grueso, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early,+but+not+late+onset+estrogen+replacement+therapy+prevents+oxidative+stress+and+metabolic+alterations+caused+by+ovariectomy.">Early, but not late onset estrogen replacement therapy prevents oxidative stress and metabolic alterations caused by ovariectomy.</a> Antioxidants and Redox Signaling. 20 (2), 236-246 (2014).</li><li>Shughrue, P. J., Bushnell, C. D., Dorsa, D. 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The authors of this manuscript have no competing financial interests or other conflicts of interest to disclose.

Fear extinction has been studied extensively in male rats with the neural mechanisms mediating conditioned fear extinction identified and assessed under various manipulations. Relatively few studies have examined female rats or the role of gonadal hormones in fear extinction. To specifically study the effects of estrogen on fear extinction, naturally cycling female rats are subjected to a three-day behavioral paradigm. This procedure consists of habituation/conditioning, extinction, and extinction recall phases. Using th...

Inherent sex differences are observed across various cognitive behaviors and learning paradigms in both humans and rodents. For example, it has been reported that women generally have stronger verbal and detail-oriented abilities, whereas men have better spatial abilities1-3. These sex differences may be due in part to the influence of gonadal hormones. High estradiol levels improve performance on tasks in which women are better, but worsen performance on tasks that men typically do well4-6. Although this evidence is compelling, the limited ability to completely control experimental environments in human studies makes it difficult to determine if these effects on behavior can be attributed specifically to hormones. Animal studies, in contrast, allow for fully controlled circumstances.
Although the basic neural mechanisms of fear extinction have been identified and are well studied in males, it is unclear whether these systems are the same in females or how they change across the estrous cycle. Fear conditioning and extinction are widely used behavioral paradigms in rodents and humans to conduct studies related to anxiety disorders. Given that women have a higher risk for anxiety disorders as well as higher symptom duration and severity7-13, it is critical to include females in these studies. The underrepresentation of females in this research may be attributed to the challenges of estrous phase monitoring and accounting for the behavioral effects of sex hormones. Laboratories that do examine females in this regard encounter issues that often go unreported or are not described in the methods for their studies.
Emerging evidence in rodents suggests that sex differences in fear extinction are modulated by gonadal hormones14-18. In fear conditioning, animals are trained to fear a particular stimulus. After a number of unreinforced presentations of the stimulus, the animals learn not to fear the cue, a process called extinction. How well the animal learns and consolidates the memory of learning this task can be observed during an extinction recall test given some time after the extinction training. Less fear expression during extinction recall demonstrates good extinction memory consolidation. Recent findings from our laboratory suggest that estrogen can modulate the consolidation of fear extinction memory and improve extinction recall. Specifically, female rats that are extinguished in proestrus, the phase of the estrous cycle during which circulating estrogen levels peak, exhibit enhanced retention of extinction memory. In contrast, females that are extinguished in the low-estrogen metestrus phase display relatively poor extinction recall, which can be improved with exogenous estradiol administration prior to or immediately after extinction training15,16. The neural mechanisms of extinction memory consolidation (including the role of gonadal hormones) in females are not clear.
In laboratory animals, the role of hormones can be investigated using invasive surgical removal procedures such as castration and ovariectomy. Subsequent to recovery from surgery, gonadal hormones are often exogenously manipulated during the performance of a behavioral task19. This approach has provided critical information about sex hormones and is useful because it allows for well-controlled manipulations of gonadal hormones (in terms of timing and dose)20-23. This approach, however, does not assess the influence of the naturally occurring fluctuations that occur across the estrous cycle nor do they represent &#8220;normal&#8221; animals, thereby limiting the translational potential to human studies. It has been well-documented that female sex hormone levels peak and decline at specific phases of the estrous cycle, and estrogen receptor expression changes within the cycle, and after ovariectomy24. Thus, there is a need to conduct studies on females with intact gonads and refine experimental designs to reliably study the effects that high and low estrogen states may have on females throughout their lifespan.
This protocol focuses on the effect of estrogen on the neurobiological systems involved in fear extinction. It describes how to: 1) carefully monitor the estrous cycle, 2) prepare effective doses of estradiol for systemic administration, and 3) follow a behavioral paradigm that includes fear conditioning, extinction, and recall in naturally cycling female rats. This protocol can be modified with other pharmacological manipulations and cellular and molecular tools to aid studies to better understand sex differences that are observed in conditioned fear extinction behavior. Note that the procedures described below are those utilized within our laboratory, and there exist a number of variations of these procedures in the literature.

<table border="0" cellpadding="0" cellspacing="0" width="613">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:120px;">Name</td>
			<td style="width:159px;">Company</td>
			<td style="width:135px;">Catalog number</td>
			<td style="width:199px;">Comments</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:120px;">Fear conditioning chamber</td>
			<td style="width:159px;">Coulbourn Instruments</td>
			<td style="width:135px;"></td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:120px;">Graphic State</td>
			<td style="width:159px;">Coulbourn Instruments</td>
			<td style="width:135px;"></td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:120px;">Sound-attenuating box</td>
			<td style="width:159px;">Med Associates, Inc.</td>
			<td style="width:135px;">NIR-022MD</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:120px;">Estradiol</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:135px;">E1024</td>
			<td style="width:199px;">In sesame oil for subcutaneous injection</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:120px;">Sesame oil</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:135px;">S3547-250ML</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:120px;">Freezescan</td>
			<td style="width:159px;">Cleversys, Inc.</td>
			<td style="width:135px;"></td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:120px;">Dip quick stain</td>
			<td style="width:159px;">Jorgensen Laboratories, Inc.</td>
			<td style="width:135px;">J0322A1, J0322A2, J0322A3</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:120px;">Cotton-tipped applicators</td>
			<td style="width:159px;">Fisher Scientific</td>
			<td style="width:135px;">23-400-114</td>
			<td style="width:199px;">6-inch, sterile</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:120px;">0.9% saline</td>
			<td style="width:159px;">LabChem, Inc.</td>
			<td style="width:135px;">LC23460-2</td>
			<td style="width:199px;">Sodium chloride w/v</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:120px;">Selectfrost microscope slides</td>
			<td style="width:159px;">Fisher Scientific</td>
			<td style="width:135px;">12-550-003</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:120px;">Virex II 256</td>
			<td style="width:159px;">Diversey, Inc.</td>
			<td style="width:135px;">5019317</td>
			<td style="width:199px;">Dilute 1:256 with water</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:120px;">Luer-Lok Tip 1ml Syringes</td>
			<td style="width:159px;">Becton Dickinson</td>
			<td style="width:135px;">309628</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:120px;">Hypodermic disposable needles</td>
			<td style="width:159px;">Exelint International, Co.</td>
			<td style="width:135px;">26402</td>
			<td style="width:199px;">26-gauge</td>
		</tr>
	</tbody>
</table>

protocol for studying extinction of conditioned fear in naturally cycling female rats

Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that neural mechanisms for certain behavioral tasks and response behaviors are different in females and males. Using females in research studies can represent a challenge because of the variation of gonadal hormones during their estrous cycle. This protocol describes well-established procedures that are useful in investigating the role of estrogen in fear extinction memory consolidation in female rats. Phase of the estrous cycle and exogenous estrogen administration prior to extinction training can influence extinction recall 24 hr later. The vaginal swabbing technique for estrous phase identification described here aids the examination and manipulation of naturally cycling gonadal hormones. The use of this basic rodent model may further delineate the mechanisms by which estrogen can modulate fear extinction memory in females.

In this fear extinction recall protocol, percent freezing was measured as an indicator of fear. Animals that extinguished well and retained the memory of the extinction training exhibited low fear on the last day of the behavioral testing during extinction recall. Male and female rats do not significantly differ in conditioned fear expression during the conditioning, extinction, and recall phases (Figure 2). However, a sex difference becomes evident when the animals are analyzed separately as high- and l...

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Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats

Gonadal hormones such as estrogen modulate memory formation in a number of experimental paradigms including fear extinction memory. This protocol describes a set of methods for investigating the influence of gonadal hormones specifically during extinction in naturally cycling females, including estrous cycle monitoring and exogenous estrogen administration.

Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that ...

protocol-for-studying-extinction-conditioned-fear-naturally-cycling

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13.0K Görüntüleme.  Massachusetts General Hospital. Gonadal hormones such as estrogen modulate memory formation in a number of experimental paradigms including fear extinction memory. This protocol describes a set of methods for investigating the influence of gonadal hormones specifically during extinction in naturally cycling females, including estrous cycle monitoring and exogenous estrogen administration.