Neurobiological and Behavioral Characterization of Adult Male and Female Mice in Prolonged Social Isolation

As social animals, our health depends on interactions with other human beings to satisfy certain natural needs. Yearly, millions suffer from chronic social isolation (SI), including those living in nursing/assisted living facilities and their caregivers. SI also manifests in our criminal justice system, where more than 80,000 people in the United States are housed in solitary confinement (SC). SC is defined as the condition where people are confined individually in a prison cell—apart from the general prison population—for between twenty-two and twenty-four hours every day on weekdays and forty-eight hours straight on weekends for a period longer than 15 days. This condition is known to have toxic physiological effects, including sleep disruption, dysregulation in the hypothalamic-pituitary-adrenal (HPA) system, increased inflammation, elevated vascular resistance and blood pressure, diminished immunity. It also has been shown to have psychological consequences, including depression, stress, cognitive deficits, memory loss, and impaired concentration. Despite these well-known consequences, we know little about the impact of SI on the brain itself. In this dissertation, I have developed a mouse model of SC in which animals (C57Bl/6J mice) are born and raised in an enriched environment (EE) and then, as adults (4 months of age), are placed into isolated conditions. After 1 or 3 months of isolation, I examined the shape, size, arborization, and spine density of neurons in specific regions of the brain selected because of their relevance to known psychological effects induced by SC including, alterations in memory (hippocampus), loss of sensory threshold and discrimination (somatosensory cortex), and deficits in motor function (motor cortex). I used a modified Golgi-Cox method to identify single neurons and then confocal microscopy and Neurolucida 360 image analysis to determine the total dendritic length per neuron, spine counts, the total neuronal volume, and the extent of dendritic branching. Additionally, I used High Performance Liquid Chromatography (HPLC) methodology to examine quantitatively alterations in the levels of the neurotransmitters norepinephrine (NE), dopamine (DA), and serotonin (5-hydroxytryptamine, 5-HT) together with their metabolites in the frontal cortex and striatum of isolated mice. Moreover, I used the rapidenzyme-linked immunosorbent assay (ELISA) method to determine the levels of brain-derived neurotrophic factor (BDNF) in the motor cortex and hippocampus of mice following their isolation. I also assessed behavioral abnormalities in isolated mice using a battery of behavioral tests, including Open Field Test (OFT) for locomotion and anxiety-related behaviors, TailSuspension Test (TST) for depression-related behaviors, Resident-Intruder Test (RIT) for social aggression, and Barnes Maze (BM) for spatial and reversal learning and memory. In addition, I also investigated the effects of 1 month of EE resocialization in 3-month isolated female mice or 1 month of exercise in 3-month isolated male mice on isolation-induced biochemical and behavioral changes. In this body of work, I found that female and male mice born and raised in EE and isolated for 1-or 3 months beginning at 4 months of age had significant alterations in the neuronal morphology of the layer II of the somatosensory cortex, layer V of the motor cortex, and the CA1 region of the rostral hippocampus. Neurons in layer II of the somatosensory cortex of 1-month isolated female mice had reduced volume, total neurite process length, dendritic branching complexity, and total estimated dendritic spine number, and increased dendritic spine density. After 3 months of isolation, the changes observed in 1 month of isolation normalized to those observed in age-matched control mice raised in EE apart from the decrease in the dendritic branching complexity, which persisted after 3 months of isolation. In male mice, neurons in layer II exhibited an increase in their dendritic spine density after 1- or 3-month of isolation. On the other hand, pyramidal neurons in layer V of the motor cortex of 1-month isolated female mice showed an increase in total process length, dendritic spine density, and total dendritic spines. After 3 months of isolation, neurons in this region showed similar morphology to those observed after 1-month of isolation with the exception that their dendritic branching became more complex. In male mice, pyramidal neurons in layer V showed a reduction in total process length and dendritic complexity after 3 months of isolation. Finally, pyramidal neurons in the CA1 region of the hippocampus of 1-month isolated female mice showed an increase in total process length as well as a reduction in dendritic spine density. After 3 months of isolation, neurons in this region showed an increase in volume, total process length, dendritic complexity, and total dendritic spine, yet normalized dendritic spine density. In male mice, pyramidal neurons in the CA1 region displayed an increase in volume, yet reduction in total process length and dendritic complexity. After 3 months of isolation, pyramidal neurons in this region showed a similar reduction in total process length and dendritic complexity together with an increase in dendritic spine density. The volume of these neurons, however, appeared to normalize after 3 months of isolation. Biochemically, 1-month isolated female mice had an increase in the level of NE and a decrease in the level of DA metabolite 3-methoxytyramine (3-MT) in the striatum. In addition, 1- month isolated female mice had alterations in their 5-HT and DA systems including, decreases in the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA) and 5-HT turnover (estimated as the 5-HIAA/5-HT ratio), and a decrease in the DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and 3-MT in the frontal cortex. After 3 months of isolation in female mice, the changes in the NE and DA systems in the striatum observed at 1 month of isolation returned to normal, yet there was an increase in the level of 5-HT in the striatum. The frontal cortex of 3-month isolated female mice, on the other hand, showed decreased NE, increased 5-HIAA and 5-HT turnover, increased DA, DOPAC, homovanillic acid (HVA) and 3-MT, and decreased DA turnover (estimated as (DOPAC+HVA)/DA). However, both 1- or 3-month isolated female mice did not show any change in the level of BDNF in the motor cortex or hippocampus. In male mice, 1 month of isolation induced no alteration in the NE system of the striatum or frontal cortices. In contrast, there were alterations in the 5-HT system in the frontal cortex including a reduction in 5-HIAA and 5-HT turnover. There were also alterations in the DA system including increases in the level of DA and 3-MT and decreases in DA turnover of the striatum. Moreover, 1-month isolated male mice showed a decrease in the level of BDNF in the motor cortex. After 3-months of isolation, male mice did not show any alterations in the NE, 5-HT, and DA systems in the striatum. However, there was an increase in 5-HT turnover in this brain region. Furthermore, the level of BDNF in 3-month isolated male mice increased in the motor cortex yet decreased in the hippocampus. Behaviorally, 1 month of isolation in female mice induced an increase in locomotor activity. 3 months of isolation in female mice induced depression-like behaviors but improved reversal memory. In male mice, 1 month of isolation led to increased locomotor activity and aggression and enhanced spatial and reversal learning and memory. After 3 months of isolation, male mice showed increased depression-related behaviors and aggression, and enhanced spatial learning. With regards to rehabilitation, I found that 1 month of resocialization in EE reversed the change in the levels of NE and 5-HT in the striatum observed in 3-month isolated female mice. In addition, it also ameliorated the changes in NE and 5-HT and normali..