Top-Down Control of Serotonergic Systems in Socioaffective Choices and Depression-Like Behaviors

Regulation of social behaviors is necessary to achieve social inclusion, establish relationships and sustain those relationships through adversity. Impairments in socio-emotional function and competence are prominent and debilitating features of major depression, yet are not traditionally recognized as cardinal symptoms of the disease. However, these deficits often persist in patients whose other mood symptoms have remitted and can predict risk of relapse, indicating an important role as a vulnerability factor. Understanding the neurobiology of socioaffective dysfunction in depression is thus important for determining the pathology of the disorder and developing effective treatments. Human imaging studies of depressive patients have consistently reported abnormal activity in the ventromedial prefrontal cortex (vmPFC), an area important for emotional processing and social cognition. Tracing studies in animals and tractography in humans have shown that the dorsal raphe nucleus (DRN) is a major projection target of the vmPFC. The DRN contains the most serotonin (5-HT) producing neurons in the brain and its output has been shown to regulate behaviors along an affiliative-agonistic axis, however it is neuronally heterogeneous. This thesis investigated the cytoarchitecture of the vmPFC-DRN microcircuit and its relevance to socioaffective behaviors using genetic mapping, whole cell electrophysiology and optogenetics. I showed that GABAergic neurons, which are the primary non-serotonergic neuronal population in the DRN, mediated top-down projections from the vmPFC onto mood-regulating 5-HT neurons and demonstrated the relevance of this pathway in mediating socioaffective decisions using the chronic social defeat stress (CSDS) paradigm. In addition, I used deep brain stimulation of the vmPFC as an antidepressant model to show that therapeutic response may rely on restoring the excitatory/inhibitory balance of inputs to 5-HT neurons. Together, these results will provide a better understanding of socioaffective circuitry and could lead to the development of more effective and efficient strategies to treat mood disorders

Q&A: How can advances in tissue clearing and optogenetics contribute to our understanding of normal and diseased biology?

Mammalian organs comprise a variety of cells that interact with each other and have distinct biological roles. Access to evaluate and perturb intact biological systems at the cellular and molecular levels is essential to fully understand their functioning in normal and diseased conditions, yet technical limitations have constrained most research to small pieces of tissue. Tissue clearing and optogenetics can help overcome this hurdle: tissue clearing affords optical interrogation of whole organs at the molecular level, and optogenetics enables the scalable control and measurement of cellular activity with light. In this Q&A, we delineate recent advances and practical challenges associated with these two techniques when applied body-wide

Gut-seeded α-synuclein fibrils promote gut dysfunction and brain pathology specifically in aged mice

Parkinson’s disease is a synucleinopathy that is characterized by motor dysfunction, death of midbrain dopaminergic neurons and accumulation of α-synuclein (α-Syn) aggregates. Evidence suggests that α-Syn aggregation can originate in peripheral tissues and progress to the brain via autonomic fibers. We tested this by inoculating the duodenal wall of mice with α-Syn preformed fibrils. Following inoculation, we observed gastrointestinal deficits and physiological changes to the enteric nervous system. Using the AAV-PHP.S capsid to target the lysosomal enzyme glucocerebrosidase for peripheral gene transfer, we found that α-Syn pathology is reduced due to the increased expression of this protein. Lastly, inoculation of α-Syn fibrils in aged mice, but not younger mice, resulted in progression of α-Syn histopathology to the midbrain and subsequent motor defects. Our results characterize peripheral synucleinopathy in prodromal Parkinson’s disease and explore cellular mechanisms for the gut-to-brain progression of α-Syn pathology

Q&A: How can advances in tissue clearing and optogenetics contribute to our understanding of normal and diseased biology?

Mammalian organs comprise a variety of cells that interact with each other and have distinct biological roles. Access to evaluate and perturb intact biological systems at the cellular and molecular levels is essential to fully understand their functioning in normal and diseased conditions, yet technical limitations have constrained most research to small pieces of tissue. Tissue clearing and optogenetics can help overcome this hurdle: tissue clearing affords optical interrogation of whole organs at the molecular level, and optogenetics enables the scalable control and measurement of cellular activity with light. In this Q&A, we delineate recent advances and practical challenges associated with these two techniques when applied body-wide

Multicolor sparse viral labeling and 3D digital tracing of enteric plexus in mouse proximal colon using a novel adeno‐associated virus capsid

Background. Intravenous administration of adeno‐associated virus (AAV) can be used as a noninvasive approach to trace neuronal morphology and links. AAV‐PHP.S is a variant of AAV9 that effectively transduces the peripheral nervous system. The objective was to label randomly and sparsely enteric plexus in the mouse colon using AAV‐PHP.S with a tunable two‐component multicolor vector system and digitally trace individual neurons and nerve fibers within microcircuits in three dimensions (3D). Methods. A vector system including a tetracycline inducer with a tet‐responsive element driving three separate fluorophores was packaged in the AAV‐PHP.S capsid. The vectors were injected retro‐orbitally in mice, and the colon was harvested 3 weeks after. Confocal microscopic images of enteric plexus were digitally segmented and traced in 3D using Neurolucida 360, neuTube, or Imaris software. Key Results. The transduction of multicolor AAV vectors induced random sparse spectral labeling of soma and neurites primarily in the myenteric plexus of the proximal colon, while neurons in the submucosal plexus were occasionally transduced. Digital tracing in 3D showed various types of wiring, including multiple conjunctions of one neuron with other neurons, neurites en route, and endings; clusters of neurons in close apposition between each other; axon–axon parallel conjunctions; and intraganglionic nerve endings consisting of multiple nerve endings and passing fibers. Most of digitally traced neuronal somas were of small or medium in size. Conclusions & Inferences. The multicolor AAV‐PHP.S‐packaged vectors enabled random sparse spectral labeling and revealed complexities of enteric microcircuit in the mouse proximal colon. The techniques can facilitate digital modeling of enteric micro‐circuitry

Multicolor sparse viral labeling and 3D digital tracing of enteric plexus in mouse proximal colon using a novel adeno‐associated virus capsid

Background. Intravenous administration of adeno‐associated virus (AAV) can be used as a noninvasive approach to trace neuronal morphology and links. AAV‐PHP.S is a variant of AAV9 that effectively transduces the peripheral nervous system. The objective was to label randomly and sparsely enteric plexus in the mouse colon using AAV‐PHP.S with a tunable two‐component multicolor vector system and digitally trace individual neurons and nerve fibers within microcircuits in three dimensions (3D). Methods. A vector system including a tetracycline inducer with a tet‐responsive element driving three separate fluorophores was packaged in the AAV‐PHP.S capsid. The vectors were injected retro‐orbitally in mice, and the colon was harvested 3 weeks after. Confocal microscopic images of enteric plexus were digitally segmented and traced in 3D using Neurolucida 360, neuTube, or Imaris software. Key Results. The transduction of multicolor AAV vectors induced random sparse spectral labeling of soma and neurites primarily in the myenteric plexus of the proximal colon, while neurons in the submucosal plexus were occasionally transduced. Digital tracing in 3D showed various types of wiring, including multiple conjunctions of one neuron with other neurons, neurites en route, and endings; clusters of neurons in close apposition between each other; axon–axon parallel conjunctions; and intraganglionic nerve endings consisting of multiple nerve endings and passing fibers. Most of digitally traced neuronal somas were of small or medium in size. Conclusions & Inferences. The multicolor AAV‐PHP.S‐packaged vectors enabled random sparse spectral labeling and revealed complexities of enteric microcircuit in the mouse proximal colon. The techniques can facilitate digital modeling of enteric micro‐circuitry

Plant functional traits differ in adaptability and are predicted to be differentially affected by climate change

1. Climate change is testing the resilience of forests worldwide pushing physiological tolerance to climatic extremes. Plant functional traits have been shown to be adapted to climate and have evolved patterns of trait correlations (similar patterns of distribution) and coordinations (mechanistic trade-off). We predicted that traits would differentiate between populations associated with climatic gradients, suggestive of adaptive variation, and correlated traits would adapt to future climate scenarios in similar ways. 2. We measured genetically determined trait variation and described patterns of correlation for seven traits: photochemical reflectance index (PRI), normalized difference vegetation index (NDVI), leaf size (LS), specific leaf area (SLA), δ13C (integrated water-use efficiency, WUE), nitrogen concentration (NCONC), and wood density (WD). All measures were conducted in an experimental plantation on 960 trees sourced from 12 populations of a key forest canopy species in southwestern Australia. 3. Significant differences were found between populations for all traits. Narrow sense heritability was significant for five traits (0.15–0.21), indicating that natural selection can drive differentiation; however, SLA (0.08) and PRI (0.11) were not significantly heritable. Generalized additive models predicted trait values across the landscape for current and future climatic conditions (>90% variance). The percent change differed markedly among traits between current and future predictions (differing as little as 1.5% (δ13C) or as much as 30% (PRI)). Some trait correlations were predicted to break down in the future (SLA:NCONC, δ13C:PRI, and NCONC:WD). 4. Synthesis: Our results suggest that traits have contrasting genotypic patterns and will be subjected to different climate selection pressures, which may lower the working optimum for functional traits. Further, traits are independently associated with different climate factors, indicating that some trait correlations may be disrupted in the future. Genetic constraints and trait correlations may limit the ability for functional traits to adapt to climate change

HDAC6 Regulates Glucocorticoid Receptor Signaling in Serotonin Pathways with Critical Impact on Stress Resilience

Genetic variations in certain components of the glucocorticoid receptor (GR) chaperone complex have been associated with the development of stress-related affective disorders and individual variability in therapeutic responses to antidepressants. Mechanisms that link GR chaperoning and stress susceptibility are not well understood. Here, we show that the effects of glucocorticoid hormones on socioaffective behaviors are critically regulated via reversible acetylation of Hsp90, a key component of the GR chaperone complex. We provide pharmacological and genetic evidence indicating that the cytoplasmic lysine deacetylase HDAC6 controls Hsp90 acetylation in the brain, and thereby modulates Hsp90–GR protein–protein interactions, as well as hormone- and stress-induced GR translocation, with a critical impact on GR downstream signaling and behavior. Pet1-Cre-driven deletion of HDAC6 in serotonin neurons, the densest HDAC6-expressing cell group in the mouse brain, dramatically reduced acute anxiogenic effects of the glucocorticoid hormone corticosterone in the open-field, elevated plus maze, and social interaction tests. Serotonin-selective depletion of HDAC6 also blocked the expression of social avoidance in mice exposed to chronic social defeat and concurrently prevented the electrophysiological and morphological changes induced, in serotonin neurons, by this murine model of traumatic stress. Together, these results identify HDAC6 inhibition as a potential new strategy for proresilience and antidepressant interventions through regulation of the Hsp90–GR heterocomplex and focal prevention of GR signaling in serotonin pathways. Our data thus uncover an alternate mechanism by which pan-HDAC inhibitors may regulate stress-related behaviors independently of their action on histones

Systemic AAV vectors for widespread and targeted gene delivery in rodents

We recently developed adeno-associated virus (AAV) capsids to facilitate efficient and noninvasive gene transfer to the central and peripheral nervous systems. However, a detailed protocol for generating and systemically delivering novel AAV variants was not previously available. In this protocol, we describe how to produce and intravenously administer AAVs to adult mice to specifically label and/or genetically manipulate cells in the nervous system and organs, including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans 8 d, excluding the time required to assess gene expression, and can be readily adopted by researchers with basic molecular biology, cell culture, and animal work experience. We provide guidelines for experimental design and choice of the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing