Techniques
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In Vivo Calcium Imaging with Miniature Microscopes
We use head-mounted miniature microendoscopes (Inscopix) to visualize the activity of individual cells within large neural ensembles during freely moving behavior. We use the activity dependent calcium sensor, GCamp, to label neurons in brain regions of interest either through viral-mediated gene delivery or through the use of transgenic mouse lines. We then implant gradient refractory index (GRIN) lenses into the mouse brain, which allows us to image the same ensemble of neurons longitudinally over several days and weeks. With this powerful technique, we can study neural network dynamics of genetically defined subpopulations of neurons in the brain of live and awake mice during the experience of stress or during cognitive or emotional behavioral tasks with unprecedented temporal resolution even in deep brain regions, such as the hippocampus.
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In Vivo Fiber Photometry
We use in vivo fiber photometry in combination with the activity dependent calcium sensor, GCamp, to measure neural population activity in the brain of freely moving mice. This technique allows us to measure activity in two different brain regions at the same time, to help us understand how different brain regions are implicated in the same behaviors, and how different brain regions react to the same environmental challenges or pharmacological treatments. Using dual color imaging, we can also visualize two different fluorophores within one brain region to study how different cell types within the same region are implicated in behavior. We also use this technique to image novel biosensors for serotonin in freely moving mice to understand how serotonin signaling in specific brain regions is affected by stress.
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Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)
DREADDs are genetically engineered human muscarinic receptors that no longer bind to their natural ligand, acetylcholine, but that are instead activated by the synthetic drug, clozapine-N-oxide (CNO). DREADDs can be inhibitory (hM4Di) or excitatory (hM3Dq) receptors that allow us to chronically silence or stimulate neural populations, respectively. We use viral-mediated delivery or transgenic mouse lines to express DREADDs in specific brain regions, in specific cell populations, or in specific projection neurons, to functionally manipulate the activity of these neurons during the experience of stress or during cognitive or emotional behavioral tasks. This approach allows us to investigate a functional role for specific cells and circuits for stress responses and behavior.
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Neural Circuit Manipulation
We use anterograde and retrograde transported viruses in combination with the Cre/loxP system to label and functionally manipulate specific connections between different brain regions. Injecting a retrogradely transported Cre virus in the prefrontal cortex, and a Cre-dependent inhibitory DREADD virus in the hippocampus allows for the specific labeling of projection neurons from the hippocampus to the prefrontal cortex with the inhibitory hM4Di receptor. After 8 weeks of virus expression, CNO can be injected into the animal or directly delivered into the brain to specifically inhibit the activity of hippocampus-PFC projection neurons during the experience of stress or during behavioral tasks to investigate the functional role of these projection neurons for stress coping and behavior.
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Next Generation Sequencing
We use next generation sequencing, such as RNA-seq or Whole Genome Bisulfite Sequencing (WGBS) to investigate gene expression and DNA methylation (DNAm) changes, respectively. These techniques allow us to gain a comprehensive understanding of how DNAm and gene expression in specific brain regions change in response to stress, how long-lasting these changes are, and if they can be transmitted from one generation to the next, and if we can find treatments to reverse these epigenetic and transcriptional responses to stress.
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Social Defeat Model
In the social defeat model, an experimental intruder mouse is paired with an aggressive resident CD1 mouse for 5 min, during which the aggressive mouse will attack and physically defeat the experimental mouse. After these 5 min of physical defeat, the experimental mouse is separated from the aggressive mouse with a perforated plexiglass divider and co-housed in the same cage for 24 hours. This paradigm is repeated for 10 days, during which the experimental mouse is paired with, and defeated by, a new aggressor every day. This model produces robust social avoidance and anxiety-like behavior in the defeat mice, and is very effective in determining mice that are susceptible or resilient to chronic stress. We have previously identified a number of brain regions that are differentially affected by this stress model in susceptible and resilient mice (Anacker et al., 2016), and we have shown that increased levels of adult hippocampal neurogenesis can confer resilience to social defeat (Anacker et al., 2018).
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Early Life Stress Models
We use the Limited Bedding and Nesting (LBN) model of early life stress to induce maternal distress and fragmented maternal care. Dams and their litters are placed on a wire mesh grid with only one third of their usual bedding and nesting material during the first week of life. Previous research has shown that this deprived housing condition causes pronounced distress to the dam and her litter, resulting in unpredictable and erratic maternal care. This distress during early life has potent and long-lasting effects on brain function and behavior of the offspring. We use this model to investigate how stress early in life affects the development of neural circuits involved in stress coping, cognition, and emotional behavior.
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Chronic Corticosterone Model
Chronic stress has repeatedly been associated with disturbances in the neuroendocrine system, particularly in the function of the Hypothalamus-Pituitary-Adrenal (HPA) axis. This impairment in HPA axis function results in dysregulation of the stress hormone cortisol in humans (corticosterone in rodents). We have previously shown that high levels of stress hormones are detrimental to neural development (Anacker et al., 2012; Anacker et al., 2013). In rodents, we can directly investigate the effects of high stress hormone levels by chronically administering corticosterone for 6 to 8 weeks in the drinking water. Chronic corticosterone administration results in pronounced anxiety-like and depressive-like behavior in mice, dysregulation of stress hormone receptor expression, and impairments in the function and development of stress-sensitive brain regions, such as the hippocampus and prefrontal cortex.
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Cognitive Flexibility Tasks
The 2-Choice Digging Task assesses reversal learning and attentional set shifting as complementary components of cognitive flexibility. In this test, mice are trained to dig in two baited bowls. During Acquisition, one bowl is baited with reward and marked with strawberry scent, while the unbaited bowl is marked with coconut scent. Mice are considered to have learned the association after 8 correct out of 10 consecutive trials. To test Compound Discrimination, the previously irrelevant dimension (digging medium) is varied but the original dimension (strawberry scent) continues to predict reward. During Reversal Learning trials, the previously unrewarded stimulus within a dimension (i.e., coconut scent), will predict reward. After Reversal Learning, mice are tested on Intradimensional Rule Shift, in which the previously rewarded dimension (odor) will be changed (e.g., from strawberry & coconut to lemon & vanilla) and one of the new odors (e.g., lemon) will predict reward. During Extradimensional Rule Shift, the stimulus of the previously irrelevant dimension (one of the two digging media) predicts reward.
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Contextual Fear Conditioning
We use contextual fear conditioning to assess fear acquisition, fear memory, fear generalization, and fear extinction. On day 1, mice are exposed to context A (rectangular walls, lemon scent, bright light) and receive 1 shock (0.75 mA, 2s) after 3 minutes. On day 2, mice are placed back in context A for 3 minutes without a shock, and freezing behavior is measured as an index of fear expression/ fear memory. Two hours later, mice are placed in a neutral context B (round walls, anis scent, red light) for 3 minutes. Freezing in the neutral context is then used to calculate fear generalization. Mice are then repeatedly exposed to context A for 3 more days without a shock, and freezing on day 5 is used as an index of fear extinction.