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

The overall goal of this procedure is to examine the effects of gonadal hormones on fear extinction behavior in females during low and high hormonal states of the Esra cycle and the role of estradiol when it is administered exogenously. This is accomplished by first monitoring the esra cycle of adult female rats and identifying which phase they are in prior to experimentation. Next, the animals that are in the estrus phase are placed into their assigned chambers for habitation and fear conditioning.

The next day when the animals are in estrous, the females are injected with estradiol or vehicle and undergo fear extinction training. Finally, on day three, the animals are placed back into the same chamber to test recall of the extinction session the day before. Ultimately, vaginal swabbing for EST strase identification allows investigators to manipulate the natural fluctuations in gonadal hormones to study their influence on fear extinction memory.

This protocol can be used to help answer key questions in the field of sex differences, such as are fear extinction processes different in males and females? And if so, what are the hormonal and neurobiological mechanisms meeting these differences? The main advantage of this protocol is that it can help promote the study of female ence in fear extinction by sharing methods to do so.

Researchers new to these methods often struggle to reliably monitor the eser cycle because the procedure can be time consuming and heavily dependent on proper technique. The implications of these studies may contribute to sex specific treatment options for fear and anxiety related disorders. This may be important given that women are twice as likely as men to develop these psychopathologies.

Lisa man, a postdoc in the lab as well as care covert. A research assistant in the lab will be showing the procedure To collect samples to monitor the estrus cycle. After preparing a 0.9%saline solution, dip a cotton tipped applicator in the liquid and use a paper towel to blot the tip of excess solution holding the tail of a rat upward and taking care not to cause distress.

Gently insert the cotton tip into the vaginal canal not too deep, and roll it around the walls to capture loose vaginal cells before swiftly. Removing the tip while not transferring too much fluid, gently roll the applicator tip onto a pre-labeled microscope slide, ensuring that the sample transfers to the slide. Collect samples for at least two complete cycles and transfer several consecutive days on one slide to be able to better track and identify daily EST extras.

Phase changes once the slides are dry, immerse them 10 times into fixative, then 10 times into staining solution. And finally, five times into counterstain solution. Rinse gently with water.

Allow the slides to air dry completely before viewing under a microscope prior to the initiation of the conditioning experiment, pre-exposed the females to the conditioning chambers with the house lights on for 20 to 30 minutes per day for three days to allow them to acclimate to the environment. Assign the box they are pre-exposed to as their chamber for the study. Thoroughly clean the boxes between sessions and animals to remove odors since they can affect behavior and EST stress cycle.

Once it is verified that the animals are in the estrus phase of the EST extra cycle, begin the habituation conditioning phase. Take pret tone baseline measurements of freezing prior to the first tone condition stimulus by measuring the seconds the animal spends a mobile during the stimulus. Absent time interval, calculate the percent freezing score by dividing the second spent immobile by the duration of the trial.

To conduct extinction training during the ME estrous phase, condition the rats in their last day of ESTRO as determined by swabbing. Place the animals into the conditioning chamber and to carry out a CS alone trial of habituation. Induce a CS tone for 30 seconds.

After waiting an average of three minutes before the next trial, repeat the CS alone trial four more times for a total of five trials prior to conditioning. Connect the foot shock grid floor to an electric stimulation generator. Use a stimulation level of 0.5 to 0.6 milliamps as the unconditioned stimulus or US and a four kilohertz 80 decibel tone as the CS immediately following the habituation session.

Begin paired CS US trials of fear conditioning by presenting a 32nd CS tone, followed by a 0.5 second US shock separated by an approximately three minute inter trial interval. Repeat the paired trial six more times at the end of the conditioning session. Return the animals to their home cages and to the animal facility until the next day on day two.

After swabbing and identifying the ME estrous phase for all animals that underwent day one of the protocol, prepare enough estradiol in sesame oil or 0.9%saline for each animal to receive a 15 micrograms per kilogram dose to administer the hormone subcutaneously. Lift the loose skin between the scapula by the neck in a gentle pinch and insert the syringe into the triangle that is formed by the skin folds. 30 minutes after the animals receive their injections, place them in the conditioning chambers and begin extinction training by performing 20 CS alone.

Trials each consisting of a 32nd four kilohertz, 80 decibel tone with approximately three minutes in between trials 24 hours later on. Day three, swab the animals that completed the sessions on days one and two after placing the animals in their assigned conditioning chambers. Begin the extinction recall session by carrying out three Cs alone trials with approximately three minutes in between trials upon completion of the recall session, return the animals to their home cages and analyze the data according to the text protocol in this fear extinction protocol.

Animals that extinguished well and retained the memory of the extinction training exhibited low fear on the last day of behavioral testing during extinction Recall. As demonstrated here, male and female rats do not differ significantly in conditioned fear expression. During each phase, a sex difference becomes evident when the animals are analyzed separately as high and low estrogen ES phase groups.

This graph shows the effect of estro phase on fear extinction memory, female rats that undergo extinction training during the low estrogen mees phase of the EST extra cycle. Do not recall the extinction memory on day three as well as those trained in PROEs. However, when metri females are subcutaneously injected with a 15 micrograms per kilogram dose of estradiol prior to extinction learning consolidation of the fear extinction memory appears to be enhanced as demonstrated by low freezing during extinction recall.

Therefore, these data indicate that gonadal hormones can modulate fear extinction recall, and also mediate sex differences in conditioned fear extinction behaviors. Additional procedures such as cannulation surgery and immunohistochemistry can be performed in conjunction with the method described here to answer questions like, where is estradiol acting in the brain to modulate fear, extinction, memory consolidation, and how is estradiol altering neuronal activity within these regions? After watching this video, You should have a clear understanding of how to study natural recycling female rats and the influence of estradiol in clear ex extension.