Impacts of early life stress on the neurobiology of depression

Methods

Summary

We will employ classical techniques to examine these questions. The behavioral assessments consist of multiple protocols frequently used in assessing depressive phenotypes in animal models of depression. Tests of learned helplessness, open field, sucrose preference and social interaction have all been used to assess depressive phenotypes in rodents. To examine cell physiology, we will take recordings (input/output, paired pulse facilitation) from brain slices in the amygdala and PFC.  Morphology will be examined by immunohistochemistry, and utilize cell counts, dendritic branching and receptor density.

Challenges

As with any novel paradigm, there are possible challenges. For this study, some of those are associated with the diversity of responses to stress in animals. Some animals may have a lower threshold for stress initially which could lead to skewed results in behavior or morphology. To combat this, we are standardizing all aspects of the study, from housing, isolation, and injection protocols, to taking samples of cortisol at multiple points of the study. A second possible challenge is accounting for sex differences, and we are taking vaginal swabs to account for estrous cycles in female mice, as well as blood level estrogen and testosterone at sacrifice. We could also see a hemispheric difference in amygdala results, and to control for that we are doing equal numbers of slices per side, as well as providing a high enough N that the differences between any groups (hemispheric, sex differences, and stress grouping) can be analyzed in detail.

Pre Analysis Plan

Our basic interest is the difference or similarity between groups based on stress exposure. This includes all behavioral assessments, electrophysiology and morphology data. We expect to demonstrate ELS as an animal model of depression that is similar to adult stress, which is a currently accepted model. Assessment of behavior will be accomplished by examining tests for tail suspension, open field, sucrose preference, and social interaction. Tail suspension examines learned helplessness measured by time immobile, open field examines anxiety measured by total locomotion time, sucrose preference examines anhedonia (lack of reinforcement) measured by amount of sucrose versus water, and social interaction examines Sociability measured by amount of time affiliating with another mouse, as well as amount of time affiliating with an unfamiliar mouse. We expect to find altered phenotypes for all tests in the ELS mouse model compared to control mice.

Assessment of neuronal function will be accomplished by examining synaptic activity and cell excitability of neurons in the amygdala and PFC. A test for synaptic activity will measure the strength of connectivity (the size of the synaptic potential) between PFC and amygdala, which is expected to be significantly reduced in the ELS model compared to control mice. A test for cell excitability will measure the magnitude of somatic spikes (i.e. action potentials), which accounts for the neuron’s capacity to generate and relay electrical information downstream the neuronal circuitry. We also expect deficient cell excitability in the amygdala and PFC in the ELS mouse model compared to control mice. 

Assessment of cell morphology will be accomplished by examining cell counts, dendritic branching and receptor density in the amygdala and PFC. Measurements of cell counts, dendritic branching and receptor density reveal the degree of network connectivity of a neuronal population. We expect these metrics to be significantly diminished in the ELS mouse model compared to control mice.

We expect all these studies to unambiguously indicate a depressive state in the ELS mouse model. However, should variability in outcomes arise, we will generate a matrix to correlate ELS and adult stress phenotypes, which we expect to show them to be significantly correlated. Interestingly, multiple outcomes, changes in behavior but not physiology for instance, will lead us to additional questions and avenues for further study to decipher the nature and impact of ELS on adult subjects.