Marble Burying Test. The Marble Burying Test is often used as a measure of anxious and compulsive behaviors, both of which alter the interactions that individuals have with drugs of abuse. Obsessive and compulsive behaviors are often seen in individuals with substance use disorders, in addition to being indicative of withdrawal. If the animal is more anxious, then it will take longer for them to approach the foreign object, a marble. Neophobia is a natural phenomenon that occurs in rodents, where they are suspicious and avoid novel objects that they interact with, for fear of danger (Xxxxxxx, 1958; Xxxxxxxxxx & Xxxxxxx, 1985). By studying the latency to approach a foreign object, we can examine the anxiolytic effects of the specific drug treatment we are studying and how it interacts with estrous status. Marble burying occurs during moments of high stress in rodents, indicating increased levels of anxiety with more marbles buried. A low level of anxiety would be represented by fewer buried marbles, indicating an anxiolytic effect. However, the most impactful information is the difference in amount of marbles buried and the latency to approach. Alterations in the processing of stressful information and the functioning of the fear learning circuit could indicate alterations in the functioning of the BLA, which is also responsible for the anxiolytic response of ethanol and allopregnanolone (Akwa et al., 1999; Xxxxxxxxx et al., 2012). This could also represent alterations in the functioning of the pathway between the BLA and mPFC, which helps regulate higher cortical processing of approach and avoid behavior.
Marble Burying Test. The Marble Burying Test (MBT) is used as a measure of neophobia and anxious/compulsive behaviors (Xxxxxxx, 1958; Xxxxxxxxxxxx & Medioni, 1985). When mice are stressed, they have a tendency to bury novel objects as a way to decrease their stress and anxiety. When in a state of ethanol-withdrawal, no sex differences were found in the animals' tendency to bury objects in the cage (Logrip et al., 2017). Another study found that presence of allopregnanolone (allo) significantly inhibited marble burying behavior as opposed to vehicle (Xxxxxx et al., 2008). Because of this, we expected there to be low numbers of marbles buried in our groups that were exposed to ethanol. To account for this, and ensure the understanding of the effects of alcohol, a group that was not exposed to any ethanol was also included. Because there was not a large difference in marbles buried within the groups exposed to alcohol (Fig. 7 a,b), we also looked at latency to approach the marble (Fig. 7, c,d). Typically, we understand short latency to approach the novel object as anxious behavior and longer latency to approach indicating a decreased interest in the novel object and an anxiolytic pattern, based on the lack of urgency in the animal in approach. However, it is possible that there are different pathways and functional activations being engaged. These hypotheses are not evident based on the numerical data, but rather the behavior of the animals seen during the behavior assay. While the increase in latency to approach the object could be representative an anxiolytic response, it could also be due to increased fear of the object. The behavior with which the animal engages the object not always represented by the approach behavior, depending on how disturbed they feel from the marble’s presence. To dissociate opposing predictions and ensure the looking at the nature of the approach/avoid behavior in males and females across ethanol exposure. Additionally, studying the mechanisms engaged in females and males could indicate alterations in functionality of the pathways, similar to what is seen in the fear learning circuit. In diestrus females and males that were not exposed to ethanol, we had a very short approach latency time, which in this case is most likely indicates high anxiety and a hesitancy to move freely in an environment with an unknown object. Estrus females approached the marble much more slowly than either the diestrus females or the males. This could indicate a potential imp...
