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

Summary

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.

Abstract

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.

Introduction

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 abilities^1-3. 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 well^4-6. Although this evidence is compelling, the limited ability to completely control experimental environments in human studies makes it difficult to determine if....

Protocol

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

1. 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. Maintain them on a diet of ad libitum access to chow and water and a 12 hr light/dark cycle. Leave rats to acclimate to the housing conditions and anima....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fear conditioning chamber	Coulbourn Instruments
Graphic State	Coulbourn Instruments
Sound-attenuating box	Med Associates, Inc.	NIR-022MD
Estradiol	Sigma-Aldrich	E1024	In sesame oil for subcutaneous injection
Sesame oil	Sigma-Aldrich	S3547-250ML
Freezescan	Cleversys, Inc.
Dip quick stain	Jorgensen Laboratories, Inc.	J0322A1, J0322A2, J0322A3
Cotton-tipped applicators	Fisher Scientific	23-400-114	6-inch, sterile
0.9% saline	LabChem, Inc.	LC23460-2	Sodium chloride w/v
Selectfrost microscope slides	Fisher Scientific	12-550-003
Virex II 256	Diversey, Inc.	5019317	Dilute 1:256 with water
Luer-Lok Tip 1ml Syringes	Becton Dickinson	309628
Hypodermic disposable needles	Exelint International, Co.	26402	26-gauge