Author(s): Nelly Zabala
Mentor(s): Jane Flinn, Cognitive Behavioral Neuroscience
Social Isolation has been found to have detrimental effects in both brain structure and function. The present study was part of a larger experiment. This study examined the neurological effects of social isolation, and whether they can be remediated after being reintroduced to a social setting. I worked alongside a Master student (Michael Lamarche) who collected the behavioral analysis data, while I collected the brain analysis data using Cresyl Violet to measure cell density and Luxol Fast Blue to measure white matter (WM) density after the behavioral data was completed. Cell density was measured in the hippocampus (HC), areas CA1, CA3 and the dentate gyrus (DG) and white matter density was measured in the corpus callosum (CC) above the HC. Imagej program was used to quantify the histological images and R software was used for statistical analysis. A One-Way Analysis of Variance (ANOVA) was used to analyze the data by taking housing condition as the independent variable, with three levels within the variable (GR, SI, SI-R). A single ANOVA was performed for cell (CA1, CA3, DG, IL, PL), and white matter density (CC). Significant cell density changes were seen in the DG but not in areas CA1 and CA3. This is consistent with the 20-week Elevated Zero Maze (EZM) behavioral analysis, from Michael Lamarche, where the isolated mice performed worse in terms of entries into the open arms as shown in and number of head dips, as reduced fear is often associated with HC atrophy among other brain regions.Also, significant changes in WM density were seen in the CC. Social reintroduction may rescue changes in cell and WM density in the DG and CC.
Slide 1
• Hi, my name is Nelly Zabala. I work in Dr. Flinn’s lab, and today I will be presenting my project on: Evaluating the Effects of Social Isolation on Social Reintroduction
• (Change slide)
Slide 2
• SI is often used to study social species. (image)
o As it allows researchers to explore the underlying mechanisms that enable sociality
• Extended periods of SI can cause behavioral impairments, such as impaired memory, social withdrawal, and increased anxiety.
• (Change slide)
Slide 3
• These symptoms are often accompanied by brain impairments. Including increased cortisol levels, neuroinflammation, atrophy of brain regions associated with social behaviors, protein loss and eventually cell density loss.
• (Change Slide)
Slide 4 – Social Enrichment
• In contrast, social enrichment has been found to lead better outcomes.
• Social enrichment refers to activities that involve social interactions, such as social group activities and social play. These behaviors help reduce isolation and promote bonding between individuals.
• In previous research. social reintroduction (SR) brought back healthy levels of hippocampal neurotrophic factors and neurogenesis.
• Now that we have talked a bit about the main topic, let’s shift our attention to some of the supporting research studies used in this project.
• (Change Slide)
Slide 5 – Human Studies
• Human studies of social isolation are limited in both intensity and duration because of the potential risks.
• However, a few studies have assessed subjective isolation (loneliness) and found significant results.
• The first one being: Wilson et al who recruited over 800 individuals and administered tests that measured loneliness and cognitive performance.
• Their findings showed that SI led to decrease cognitive function and an increased risk for dementia.
• The second study is a systematic review by Cacioppo et al. (2011), they suggested that social isolation pose as much risk for developing health problems and premature death as smoking, obesity, sedentary lifestyle, and high blood pressure.
• (Change slide)
Slide 6 – Mice Studies
• Now, there are many mice studies associated with SI (wait)
• Cao et al. who found that social isolation induced changes in both the HC and PFC, these changes were accompanied by learning and memory deficits and spatial cognitive impairments in the HC. There were also social interactions defects and anxiety in the PFC, as well as changes in protein expression.
• EE rescued these changes in the HC (shown by the solid line) but not in the PFC (shown by the dotted line). The PFC has been associated with social behaviors, demonstrating that some form of social interaction may also be needed.
• (Change Slide)
Slide 7 – Mice Studies cont.
• The next study by Liu et al demonstrated WM reduction in mice, in the form of myelination changes, which were rescued by introducing the mice to a social setting.
• Biggio et al SI Sprague-Dawley rats and found changes in the HC and decrease protein expression.
• Reintroduction rescued changes in the DG and increased protein expression.
• (Change Slide)
Slide 8 – Current Study
• This project addressed changes in brain functions to see if the detrimental effects of social isolation can be rescued by reintroduction to a social setting.
• It is based on previous research in our lab by Rachel Barkey:
• Who found that mice who were socially isolated showed disruptions in circadian rhythms when compared to mice who remained group housed.
• I worked along with a master student, Michael Lamarche who did behavioral analysis while I did biochemical analysis.
• (Change Slide)
Slide 9 – Timeline
• At 12 weeks following behavioral analysis, mice were then divided; continually group housed (GH), continually socially isolated (SI), and half of the socially isolated mice were then group housed (SI-R); thus, there were three groups.
• At 20 weeks, behavioral testing was performed again, and brains extracted for histology and western blot analysis.
• (Change Slide)
Slide 10 – Block-Party cages
• The SI-R mice were housed in block-party cages, which consisted of individual housing units connected by tubes previously blocked. This allowed the mice to get used to each other’s scent and presence.
• (Change Slide)
Slide 11 – Biochemical Measures
• After euthanasia, the brains were extracted and stored at -80 degrees.
• Cresyl violet was used to assess cell density.
• (Change Slide)
Slide 12 – Anatomical Region
â— This image shows you the anatomical regions where we measured cell and white matter density.
â— White matter density was measured above the HC in the CC
â— Cell density in the HC (CA1, CA3 and DG)
â— (Change Slide)
Slide 13 – Statistical Analysis
• Imagej program was used to quantify the western blot and histological images
• A One-Way Analysis of Variance (ANOVA) was used to analyze the data by taking housing condition as the independent variable, with three levels within the variable (GR, SI, SI-R)
• A single ANOVA was performed for cell and WM density.
• Any significant results were followed by a Tukey post hoc test to identify pairwise differences.
• (Change Slide)
Slide 14 and 15 – CA1 – CA3
• Our first hypothesis predicted an increase in cell density in the HC areas (CA1, CA3, DG) in mice who were socially reintroduced compared to those who remained isolated.
• There were no significant changes in areas CA1 and CA3 of the HC. However, a one-way ANOVA revealed a significant difference between the DG and housing condition.
• (Change Slide)
• The p-values are shown in this table.
• (Change Slide)
Slide 16 – DG
• A post hoc of the DG showed significant difference between GH and SI mice (p = 0.0128) but not between SI-R and GH or SI-R and SI
Slide 17 and 18- WM
• Our next hypothesis predicted an increase in WM density in the CC.
• A Welch’s test also revealed significant results for this region.
• (Change Slide)
• Post hoc revealed significant differences between GH and SI mice and SI and SI-R mice. There were no significant differences between GH and SI-R
• (Change Slide)
Slide 19 to 21- Discussion (DG)
• Lack of significant differences in area CA1 and CA3 could mean that social behaviors may not affect these regions as much as it does the DG, which showed significant results.
• On the other hand, DG has been associated with neurogenesis and increased susceptibility to environmental factors
• Decrease in cells density may be due to the impact that SI has on neurogenesis and anxiety.
• (Change Slide)
• This image shows evident changes in DG morphology between the GH and SI groups. While the SI-R group displays a smaller variation in morphology when compared to the GH, this variation was not significant.
• (Change Slide)
• This is consistent with the 20-week Elevated Zero Maze (EZM) behavioral analysis from Michael Lamarche, where the isolated mice performed worse in terms of entries into the open arms as shown in (Figure 8a) and number of head dips as shown in figure 8b, as reduced fear is often associated with hippocampal atrophy among other brain regions.
• (Change Slide)
Slide 22 to 24 – Discussion cont. WM
• This study also found a decrease in WM density, which can be associated with impaired neurological processes, such as learning and memory.
• The CC is responsible for carrying information between the HC and other areas of the brain.
• (Change Slide)
• Therefore, the decrease in WM density may also explain why socially isolated mice performed worse in terms of entries into the open arms and number of head dips.
• (Change Slide)
• This figure demonstrates WM density between the groups, with the WM density in SI mice being significantly different than GH and SI-R.
• Implying that reintroduction could potentially rescue the detrimental effects of SI in the CC
• (Change Slide)
Slide 25 – Future Research
• In this study, SI mice were reintroduced to previously SI mice. It could be possible that the withdrawal symptoms from isolation did not allow for recovery of social behaviors during reintroduction.
• Environmental enrichment may have provided additional benefits to the mice during SI and SI-R.
• In this study, the reintroduced mice were observed for a duration of 8 weeks. However, future research should examine a more extended period of acclimation following reintroduction.
• Hi, my name is Nelly Zabala. I work in Dr. Flinn’s lab, and today I will be presenting my project on: Evaluating the Effects of Social Isolation on Social Reintroduction
• (Change slide)
Slide 2
• SI is often used to study social species. (image)
o As it allows researchers to explore the underlying mechanisms that enable sociality
• Extended periods of SI can cause behavioral impairments, such as impaired memory, social withdrawal, and increased anxiety.
• (Change slide)
Slide 3
• These symptoms are often accompanied by brain impairments. Including increased cortisol levels, neuroinflammation, atrophy of brain regions associated with social behaviors, protein loss and eventually cell density loss.
• (Change Slide)
Slide 4 – Social Enrichment
• In contrast, social enrichment has been found to lead better outcomes.
• Social enrichment refers to activities that involve social interactions, such as social group activities and social play. These behaviors help reduce isolation and promote bonding between individuals.
• In previous research. social reintroduction (SR) brought back healthy levels of hippocampal neurotrophic factors and neurogenesis.
• Now that we have talked a bit about the main topic, let’s shift our attention to some of the supporting research studies used in this project.
• (Change Slide)
Slide 5 – Human Studies
• Human studies of social isolation are limited in both intensity and duration because of the potential risks.
• However, a few studies have assessed subjective isolation (loneliness) and found significant results.
• The first one being: Wilson et al who recruited over 800 individuals and administered tests that measured loneliness and cognitive performance.
• Their findings showed that SI led to decrease cognitive function and an increased risk for dementia.
• The second study is a systematic review by Cacioppo et al. (2011), they suggested that social isolation pose as much risk for developing health problems and premature death as smoking, obesity, sedentary lifestyle, and high blood pressure.
• (Change slide)
Slide 6 – Mice Studies
• Now, there are many mice studies associated with SI (wait)
• Cao et al. who found that social isolation induced changes in both the HC and PFC, these changes were accompanied by learning and memory deficits and spatial cognitive impairments in the HC. There were also social interactions defects and anxiety in the PFC, as well as changes in protein expression.
• EE rescued these changes in the HC (shown by the solid line) but not in the PFC (shown by the dotted line). The PFC has been associated with social behaviors, demonstrating that some form of social interaction may also be needed.
• (Change Slide)
Slide 7 – Mice Studies cont.
• The next study by Liu et al demonstrated WM reduction in mice, in the form of myelination changes, which were rescued by introducing the mice to a social setting.
• Biggio et al SI Sprague-Dawley rats and found changes in the HC and decrease protein expression.
• Reintroduction rescued changes in the DG and increased protein expression.
• (Change Slide)
Slide 8 – Current Study
• This project addressed changes in brain functions to see if the detrimental effects of social isolation can be rescued by reintroduction to a social setting.
• It is based on previous research in our lab by Rachel Barkey:
• Who found that mice who were socially isolated showed disruptions in circadian rhythms when compared to mice who remained group housed.
• I worked along with a master student, Michael Lamarche who did behavioral analysis while I did biochemical analysis.
• (Change Slide)
Slide 9 – Timeline
• At 12 weeks following behavioral analysis, mice were then divided; continually group housed (GH), continually socially isolated (SI), and half of the socially isolated mice were then group housed (SI-R); thus, there were three groups.
• At 20 weeks, behavioral testing was performed again, and brains extracted for histology and western blot analysis.
• (Change Slide)
Slide 10 – Block-Party cages
• The SI-R mice were housed in block-party cages, which consisted of individual housing units connected by tubes previously blocked. This allowed the mice to get used to each other’s scent and presence.
• (Change Slide)
Slide 11 – Biochemical Measures
• After euthanasia, the brains were extracted and stored at -80 degrees.
• Cresyl violet was used to assess cell density.
• (Change Slide)
Slide 12 – Anatomical Region
â— This image shows you the anatomical regions where we measured cell and white matter density.
â— White matter density was measured above the HC in the CC
â— Cell density in the HC (CA1, CA3 and DG)
â— (Change Slide)
Slide 13 – Statistical Analysis
• Imagej program was used to quantify the western blot and histological images
• A One-Way Analysis of Variance (ANOVA) was used to analyze the data by taking housing condition as the independent variable, with three levels within the variable (GR, SI, SI-R)
• A single ANOVA was performed for cell and WM density.
• Any significant results were followed by a Tukey post hoc test to identify pairwise differences.
• (Change Slide)
Slide 14 and 15 – CA1 – CA3
• Our first hypothesis predicted an increase in cell density in the HC areas (CA1, CA3, DG) in mice who were socially reintroduced compared to those who remained isolated.
• There were no significant changes in areas CA1 and CA3 of the HC. However, a one-way ANOVA revealed a significant difference between the DG and housing condition.
• (Change Slide)
• The p-values are shown in this table.
• (Change Slide)
Slide 16 – DG
• A post hoc of the DG showed significant difference between GH and SI mice (p = 0.0128) but not between SI-R and GH or SI-R and SI
Slide 17 and 18- WM
• Our next hypothesis predicted an increase in WM density in the CC.
• A Welch’s test also revealed significant results for this region.
• (Change Slide)
• Post hoc revealed significant differences between GH and SI mice and SI and SI-R mice. There were no significant differences between GH and SI-R
• (Change Slide)
Slide 19 to 21- Discussion (DG)
• Lack of significant differences in area CA1 and CA3 could mean that social behaviors may not affect these regions as much as it does the DG, which showed significant results.
• On the other hand, DG has been associated with neurogenesis and increased susceptibility to environmental factors
• Decrease in cells density may be due to the impact that SI has on neurogenesis and anxiety.
• (Change Slide)
• This image shows evident changes in DG morphology between the GH and SI groups. While the SI-R group displays a smaller variation in morphology when compared to the GH, this variation was not significant.
• (Change Slide)
• This is consistent with the 20-week Elevated Zero Maze (EZM) behavioral analysis from Michael Lamarche, where the isolated mice performed worse in terms of entries into the open arms as shown in (Figure 8a) and number of head dips as shown in figure 8b, as reduced fear is often associated with hippocampal atrophy among other brain regions.
• (Change Slide)
Slide 22 to 24 – Discussion cont. WM
• This study also found a decrease in WM density, which can be associated with impaired neurological processes, such as learning and memory.
• The CC is responsible for carrying information between the HC and other areas of the brain.
• (Change Slide)
• Therefore, the decrease in WM density may also explain why socially isolated mice performed worse in terms of entries into the open arms and number of head dips.
• (Change Slide)
• This figure demonstrates WM density between the groups, with the WM density in SI mice being significantly different than GH and SI-R.
• Implying that reintroduction could potentially rescue the detrimental effects of SI in the CC
• (Change Slide)
Slide 25 – Future Research
• In this study, SI mice were reintroduced to previously SI mice. It could be possible that the withdrawal symptoms from isolation did not allow for recovery of social behaviors during reintroduction.
• Environmental enrichment may have provided additional benefits to the mice during SI and SI-R.
• In this study, the reintroduced mice were observed for a duration of 8 weeks. However, future research should examine a more extended period of acclimation following reintroduction.
2 replies on “Evaluating the Effects of Social Isolation on Social Reintroduction”
Nelly, this was a great project! I was surprised to learn that social isolation can affect cognitive processes like learning and memory in addition to mood/behavioral effects. I hope you get to perform the future studies and examine a longer reintegration period so that we can better understand the longer-term effects of periods of social isolation.
Well done. I look forward to hearing more about the work. Understanding the effects of social isolation is so important.