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512. shRNA-Based Suppression of Connexin 43 and Low Packing Density of Connexin 43 Immunoreactive Aggregates are Associated with Depression-Like Behavior in Rats
Biological Psychiatry
Friday Abstracts
Supported By: RO1 Keywords: Oxidative Stress, Major Depression, Childhood Trauma, Parenting
511. Stress-Induced Neuronal CSF1 Provokes Microglia-Mediated Dendritic Remodeling and Depressive-Like Behavior Eric Wohleb1, Rosemarie Terwilliger2, Catharine Duman2, and Ronald Duman2 University of Cincinnati College of Medicine, 2Yale University School of Medicine
1
Background: Chronic stress exposure causes pyramidal neuron dystrophy and synaptic deficits in the medial prefrontal cortex (PFC), which leads to development of anxiety-and depressive-like behaviors. Concomitantly microglia in the PFC undergo morphological and functional changes following stress exposure, suggesting that microglia contribute to stress-induced synaptic deficits underlying behavioral consequences. Methods: Male and female mice were exposed to chronic unpredictable stress (CUS) to examine the effect of neuronmicroglia interactions on synaptic deficits in the medial PFC. Thy1-GFP-M were used to assess microglia-mediated dendritic remodeling and spine density of pyramidal neurons in the medial PFC. Studies using viral-mediated knockdown determined the role of neuronal colony stimulating factor-1 (CSF1) in modulating microglia function and anxiety- or depressive-like behaviors after CUS. Results: CUS promoted anxiety- and depressive-like behaviors that were associated with increased mRNA levels of CSF1 in the PFC. Increased CSF1 mRNA levels were also detected in postmortem dorsolateral PFC of depressed individuals. Moreover, frontal cortex microglia isolated from mice exposed to CUS show increased CSF1 receptor expression, morphological reactivity changes, and increased phagocytosis of neuronal elements. These functional changes in microglia corresponded with reduced dendritic spine density on pyramidal neurons in layer I of the medial PFC. Viral-mediated knockdown of neuronal CSF1 in the medial PFC attenuated microglia-mediated dendritic remodeling and prevented behavioral deficits caused by CUS. Conclusions: These findings revealed that stress-induced elevations in neuronal CSF1 provoke microglia-mediated dendritic remodeling in the medial PFC, contributing to synaptic deficits and development of anxiety- and depressive-like behavior. Supported By: NIH MH045481; NIH MH093897; State of Connecticut Keywords: Depression, Stress, Microglia, Neuroplasticity, Prefrontal Cortex
512. shRNA-Based Suppression of Connexin 43 and Low Packing Density of Connexin 43 Immunoreactive Aggregates are Associated with Depression-Like Behavior in Rats Jose Miguel-Hidalgo, Mohadetheh Moulana, Natalie Booker, and Grazyna Rajkowska University of Mississippi Medical Center
S208
Background: Background: Astrocytes and oligodendrocytes, known to be connected through gap junctions (GAPJs), are pathologically altered in the prefrontal cortex (PFC) in depression. GAPJ protein of astrocytes connexin 43 (Cx43) is dramatically reduced in the PFC of human subjects with depression and in animal models of depression-like behaviors, although there is no direct 3-dimensional evidence of reduced Cx43-immunoreactive gap junction aggregates or that localized Cx43 reduction leads to depression-like behaviors. Methods: Methods: Frozen sections from the prelimbic cortex (PLC, part of the PFC) of rats subjected to 28 days of chronic unpredictable stress (CUS, which results in depression-like behaviors) and controls were processed for immunohistochemistry of Cx43, and the 3-dimensional density of Cx43 immunoreactive puncta in the PLC was determined with StereoInvestigator. Other rats were infused in the PLC with lentiviruses either with Cx43-shRNA for Cx43 suppression or with scrambled shRNA (non-suppressing) as control, and consumption of an aqueous sucrose solution and of plain water were measured. Results: Results: 3-dimensional packing density of Cx43 immunoreactive puncta in the PLC was significantly lower in the PLC of adult rats subjected to CUS than in controls. Infusion of Cx43-shRNA resulted in a significant 60% reduction in the consumption of the sucrose solution, without change in consumption of plain water. Conclusions: Conclusion: The in situ morphometric evidence supports that the packing density of aggregates (puncta) with Cx43-positive GAPJs is reduced in the PLC of CUS-exposed rats. In addition, depletion of Cx43 appears to reduce sucrose consumption, which has been related to anhedonia, a major symptom of depression. Supported By: NIMH grant MH82297, Animal Behavior core and Imaging Core of NIGMS grant P30GM103328 Keywords: Depression, Astrocytes, gap junctions, Sucrose Preference, Unpredictable Chronic Mild Stress
513. Functional Characterization of Ankyrin Loss of Function Mutations Associated with Autism Spectrum Disorder Jacob Garza and Tracey Petryshen Massachusetts General Hospital Background: Recent large-scale genomic studies have identified candidate genes involved in synaptic transmission that are highly associated with autism spectrum disorder (ASD), including ANK2 and ANK3. Loss of function missense mutations was associated with autistic patients. The goal of this project is to functionally characterize de novo loss of function mutations in ankyrins and identify their contribution to ASD. Methods: A CRISPR/Cas9-based approach was used to transcriptionally repress ankyrin in a neuronal cell model. Fourteen sgRNA and one non-targeting control sgRNA were designed and screened for efficiency of transcriptional repression. Ankyrin levels were measured by qPCR. Tubulin polymerization and end-binding protein 3 expression were evaluated using Western blot. Furthermore, CRISPR/Cas9 was also used to induce point mutations within the Ank2 gene.
Biological Psychiatry May 15, 2017; 81:S140–S276 www.sobp.org/journal
Friday Abstracts
Supported By: RO1 Keywords: Oxidative Stress, Major Depression, Childhood Trauma, Parenting
511. Stress-Induced Neuronal CSF1 Provokes Microglia-Mediated Dendritic Remodeling and Depressive-Like Behavior Eric Wohleb1, Rosemarie Terwilliger2, Catharine Duman2, and Ronald Duman2 University of Cincinnati College of Medicine, 2Yale University School of Medicine
1
Background: Chronic stress exposure causes pyramidal neuron dystrophy and synaptic deficits in the medial prefrontal cortex (PFC), which leads to development of anxiety-and depressive-like behaviors. Concomitantly microglia in the PFC undergo morphological and functional changes following stress exposure, suggesting that microglia contribute to stress-induced synaptic deficits underlying behavioral consequences. Methods: Male and female mice were exposed to chronic unpredictable stress (CUS) to examine the effect of neuronmicroglia interactions on synaptic deficits in the medial PFC. Thy1-GFP-M were used to assess microglia-mediated dendritic remodeling and spine density of pyramidal neurons in the medial PFC. Studies using viral-mediated knockdown determined the role of neuronal colony stimulating factor-1 (CSF1) in modulating microglia function and anxiety- or depressive-like behaviors after CUS. Results: CUS promoted anxiety- and depressive-like behaviors that were associated with increased mRNA levels of CSF1 in the PFC. Increased CSF1 mRNA levels were also detected in postmortem dorsolateral PFC of depressed individuals. Moreover, frontal cortex microglia isolated from mice exposed to CUS show increased CSF1 receptor expression, morphological reactivity changes, and increased phagocytosis of neuronal elements. These functional changes in microglia corresponded with reduced dendritic spine density on pyramidal neurons in layer I of the medial PFC. Viral-mediated knockdown of neuronal CSF1 in the medial PFC attenuated microglia-mediated dendritic remodeling and prevented behavioral deficits caused by CUS. Conclusions: These findings revealed that stress-induced elevations in neuronal CSF1 provoke microglia-mediated dendritic remodeling in the medial PFC, contributing to synaptic deficits and development of anxiety- and depressive-like behavior. Supported By: NIH MH045481; NIH MH093897; State of Connecticut Keywords: Depression, Stress, Microglia, Neuroplasticity, Prefrontal Cortex
512. shRNA-Based Suppression of Connexin 43 and Low Packing Density of Connexin 43 Immunoreactive Aggregates are Associated with Depression-Like Behavior in Rats Jose Miguel-Hidalgo, Mohadetheh Moulana, Natalie Booker, and Grazyna Rajkowska University of Mississippi Medical Center
S208
Background: Background: Astrocytes and oligodendrocytes, known to be connected through gap junctions (GAPJs), are pathologically altered in the prefrontal cortex (PFC) in depression. GAPJ protein of astrocytes connexin 43 (Cx43) is dramatically reduced in the PFC of human subjects with depression and in animal models of depression-like behaviors, although there is no direct 3-dimensional evidence of reduced Cx43-immunoreactive gap junction aggregates or that localized Cx43 reduction leads to depression-like behaviors. Methods: Methods: Frozen sections from the prelimbic cortex (PLC, part of the PFC) of rats subjected to 28 days of chronic unpredictable stress (CUS, which results in depression-like behaviors) and controls were processed for immunohistochemistry of Cx43, and the 3-dimensional density of Cx43 immunoreactive puncta in the PLC was determined with StereoInvestigator. Other rats were infused in the PLC with lentiviruses either with Cx43-shRNA for Cx43 suppression or with scrambled shRNA (non-suppressing) as control, and consumption of an aqueous sucrose solution and of plain water were measured. Results: Results: 3-dimensional packing density of Cx43 immunoreactive puncta in the PLC was significantly lower in the PLC of adult rats subjected to CUS than in controls. Infusion of Cx43-shRNA resulted in a significant 60% reduction in the consumption of the sucrose solution, without change in consumption of plain water. Conclusions: Conclusion: The in situ morphometric evidence supports that the packing density of aggregates (puncta) with Cx43-positive GAPJs is reduced in the PLC of CUS-exposed rats. In addition, depletion of Cx43 appears to reduce sucrose consumption, which has been related to anhedonia, a major symptom of depression. Supported By: NIMH grant MH82297, Animal Behavior core and Imaging Core of NIGMS grant P30GM103328 Keywords: Depression, Astrocytes, gap junctions, Sucrose Preference, Unpredictable Chronic Mild Stress
513. Functional Characterization of Ankyrin Loss of Function Mutations Associated with Autism Spectrum Disorder Jacob Garza and Tracey Petryshen Massachusetts General Hospital Background: Recent large-scale genomic studies have identified candidate genes involved in synaptic transmission that are highly associated with autism spectrum disorder (ASD), including ANK2 and ANK3. Loss of function missense mutations was associated with autistic patients. The goal of this project is to functionally characterize de novo loss of function mutations in ankyrins and identify their contribution to ASD. Methods: A CRISPR/Cas9-based approach was used to transcriptionally repress ankyrin in a neuronal cell model. Fourteen sgRNA and one non-targeting control sgRNA were designed and screened for efficiency of transcriptional repression. Ankyrin levels were measured by qPCR. Tubulin polymerization and end-binding protein 3 expression were evaluated using Western blot. Furthermore, CRISPR/Cas9 was also used to induce point mutations within the Ank2 gene.
Biological Psychiatry May 15, 2017; 81:S140–S276 www.sobp.org/journal