Involvement of mu-opioid receptors in mediating the long-term impacts of early life stress

Abstract

Post traumatic stress disorder (PTSD) is a mental health condition that is caused by an extremely stressful or traumatic event. In recent years fear research has been utilized to better understand how PTSD manifests within the brain. Several studies show that mu opioid receptors are linked to fear learning. There are a large number of these receptors in the amygdala, a brain region critical for fear. We and others have shown that exposure to early adversity leads to increased fear learning in adulthood. In this study, we will address whether this “stress-enhanced fear learning” (i.e., SEFL) is mediated by how rodents mu opioid receptors in the amygdala. We will use a genetically modified line of mice that have these receptors knocked out in the amygdala (i.e., MOR-KO) and control mice. Mice will be exposed to acute early life stress (aELS) or control (no-aELS) in infancy and then undergo fear conditioning and testing in adulthood. We hypothesize that the SEFL observed in previously-stressed animals is mediated by an increase in mu-opioid receptor expression in the amygdala. Thus, knockout of these receptors should eliminate SEFL in mice previously exposed to aELS.

Introduction

In recent years, Posttraumatic stress disorder (PTSD) has gained a significant amount of attention, both in the public eye and within the medical community. Once largely associated with combat veterans, PTSD is now recognized as a mental health condition that affects people from all walks of life, including survivors of accidents, natural disasters, assaults, and other traumatic events. Females experience an increased risk of developing PTSD (National Institute of Mental Health, 2023). As of 2017, the cross-national lifetime prevalence of PTSD is 3.9% (Koenen et al., 2017). In recent research, it has been found that safety learning may play a major role in adaptive fear response (Jovanovic et al., 2012). 

In order to study fear learning and adaptive fear response without being invasive to humans it is necessary that we use animal models. This  allows us to carefully manipulate environmental factors in order to get more accurate results from our research due to the fact that there are now less variables at play in our models (Phillips & Roth, 2019). The paradigm we will be using is known as stress enhanced fear learning (SEFL). By using this model we are able to look at how extreme stress can cause long-lasting changes in affective behavior (Nishimura et al., 2022). In our study, the event of extreme stress is a slight shock administered through the floor of a box. Afterwards, the mice will be tested in the same box but without the shock for freezing to see if the fear learning has been acquired. Due to the prior shocks the mice should develop a fear response to the conditioned stimulus. We expect to see higher freezing in animals that have been conditioned with the shock and who now have developed a fear response. 

A mu opioid receptor (MOR) is a G-protein-coupled receptor (GPCR) that binds to both endogenous and exogenous opioids and is involved in many physiological functions. MOR’s have been linked to addictive and rewarding behaviors (Carvour et al., 2023). Research has shown that endogenous opioid receptors are involved in fear learning in rodents (Eippert et al., 2008). By blocking these receptors we will be able to see how much of an effect they truly have on fear learning in rodents. The amygdala is a key brain region that has been identified as an important binding site for MORs. These MORs within the amygdala appear to influence addictive behaviors when activated (Hamida et al., 2019). Within the amygdala, MOR expression is compacted in the Foxp2-expressing intercalated (ITC) cells. ITC cells are GABAergic interneurons that are critical regulators of extinction learning and are typically located around the basal lateral amygdala (Likhtik et al., 2008). We will be using a line of mice that has the mu opioid receptors bred out over many generations. This breeding is a global knockout of the mu opioid receptors, which means there are no mu opioid receptors anywhere in the brain for endogenous opioids to bind to. 

Through our experiment, we expect to find that mice who experience aELS exposure will increase MOR expression within the amygdala. Mice with the knocked out MORs will eliminate SEFL in the mice that have been previously exposed to stress. From existing research, it has been observed that male mice tend to exhibit a greater fear response compared to female mice when put through the same fear conditioning paradigms (Clark et al., 2019). Therefore, in addition with the difference between wild-type mice and mice that have mu opioid receptors knocked out, we expect that there will also be a sex difference based on preliminary data. While our preliminary data does not exhibit a genotype difference due to being underpowered, we expect to see a genotypic increase in the fear response of knockout mice following the addition of 75 mice. We hypothesize that the SEFL observed in previously-stressed animals is mediated by an increase in mu-opioid receptor expression in the amygdala. Thus, knockout of these receptors should eliminate SEFL in mice previously exposed to aELS.

Experimental design 

In this study, we will use a 2 (genotype: wildtype control vs. mu opioid receptor knock-out) X 2 (sex: male vs. female) X 2 (aELS: no-aELS vs. aELS) factorial design yielding 8 experimental conditions. We will use 14 mice/condition for a total of 112. Thus far, we have run 82 mice in this experiment. 

Animals

The animals used will be Foxp2-Cre X Oprm1-Flox mice (short handed to fMOR). This line of mice has a genetic deletion of Oprm1 in Foxp2-Cre X Oprm1-Flox neurons. This deletion will allow for the observation of the role the intercalated cells in the amygdala plays in stress-enhanced fear learning. The mice will be weaned until PND 21 with same-sex litter mates. After weaning, the mice will remain with same-sex litter mates (on average, 2 other litter mates).

Procedure 

Early life stress. Context A is an early life stress exposure that all mice are placed in on PND 17.  Context A will take place in four identical conditioning chambers (32.4 × 25.4 × 21.6 cm; Med-Associates Inc., Georgia, Vermont) within sound-attenuating cubicles. Chambers consist of a white, plastic back wall, aluminum sidewalls, a clear Plexiglas ceiling and front door. The chambers will be brightly lit (140 lux) and have a flat, stainless-steel grid floor with an underlying pan coated in approximately 10 ml of 50% vanilla odorant without bedding material (Kroger Inc.). Grid floors will be wired to a shock generator and scrambler (Med-Associates, Inc.).

During transport infant mice will not be removed from their dams and will be with littermates and dam in a plastic cage. On PND 17 the cage will be transported to the laboratory and held in a room near Context A. The mice will then be placed individually into Context A for 60 minutes and receive fear conditioning which consists of either 15 or 0 foot shocks of 1 mA for 1 second. In the 15 footshock stress exposure, the first footshock will be delivered 180 s after the mouse is placed into the chamber, and subsequent foot shocks will be delivered with a variable intershock interval of 240–480 s (average of 360 s). Zero footshock animals will be placed into the Context A chambers for the 60 minutes but will not receive any footshocks. After the stress exposure session, mice will be removed from the chamber and returned to the holding room with their littermates and dam.

Adult fear conditioning and testing. Context B will be a novel context that all mice are placed in on PND 61. This context will be in a different room from Context A. The chambers in Context B will be made of black, triangular Plexiglass inserts and floors made of 18 staggered stainless-steel rods in two rows that are 0.5 centimeters apart vertically with each rod 1.5 centimeters apart. The chambers will be completely dark (with near-infrared lighting used for video recording), and have underlying pans coated with approximately 10 milliliters of white vinegar (Kroger Inc.) to serve as a context odor. Mice will be transported between the context and the home cages in individual, blackened plastic containers (18 cm × 32 cm × 9 cm).

On PND 61, the mice will be placed in Context B for 3.5 minutes and receive fear conditioning which consists of 1 footshock of 1 mA for 1 second. On PND 62, the mice will receive a conditioned fear stimulus test in Context B for 8 minutes as a fear memory test. 

Data analysis 

In order to assess fear response in the animals during the experimental sessions we tracked their freezing behavior using cameras within the enclosure. In order to examine the main effects and relationships between sex (male/female), fMOR (knocked out/present) and early life stress exposure (0 footshocks, 15 footshocks) we used factorial analysis of variance (ANOVAs) to access the found data. A critical value α = 0.05 was utilized for all analyses. 

Future research can utilize this information to determine how amygdaloid microcircuits impact fear acquisition and expression.

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Work was approved under IACUC protocols