Oxytocin signaling is necessary for synaptic maturation of adult-born neurons.

Neural circuit plasticity and sensory response dynamics depend on forming new synaptic connections. Despite recent advances toward understanding the consequences of circuit plasticity, the mechanisms driving circuit plasticity are unknown. Adult-born neurons within the olfactory bulb have proven to be a powerful model for studying circuit plasticity, providing a broad and accessible avenue into neuron development, migration, and circuit integration. We and others have shown that efficient adult-born neuron circuit integration hinges on presynaptic activity in the form of diverse signaling peptides. Here, we demonstrate a novel oxytocin-dependent mechanism of adult-born neuron synaptic maturation and circuit integration. We reveal spatial and temporal enrichment of oxytocin receptor expression within adult-born neurons in the murine olfactory bulb, with oxytocin receptor expression peaking during activity-dependent integration. Using viral labeling, confocal microscopy, and cell type-specific RNA-seq, we demonstrate that oxytocin receptor signaling promotes synaptic maturation of newly integrating adult-born neurons by regulating their morphological development and expression of mature synaptic AMPARs and other structural proteins.

Parallel astrocyte calcium signaling modulates olfactory bulb responses.

Astrocytes are the most abundant glial cell in the central nervous system. They modulate synaptic function through a variety of mechanisms, and yet remain relatively understudied with respect to overall neuronal circuit function. Exploiting the tractability of the mouse olfactory system, we manipulated astrocyte activity and examined how astrocytes modulate olfactory bulb responses. Toward this, we genetically targeted both astrocytes and neurons for in vivo widefield imaging of Ca2+ responses to odor stimuli. We found that astrocytes exhibited odor response maps that overlap with excitatory neuronal activity. By manipulating Ca2+ activity in astrocytes using chemical genetics we found that odor-evoked neuronal activity was reciprocally affected, suggesting that astrocyte activation inhibits neuronal odor responses. Subsequently, behavioral experiments revealed that astrocyte manipulations affect both odor detection threshold and discrimination, suggesting that astrocytes play an active role in olfactory sensory processing circuits. Together, these studies show that astrocyte calcium signaling contributes to olfactory behavior through modulation of sensory circuits.

POU6f1 Mediates Neuropeptide-Dependent Plasticity in the Adult Brain.

The mouse olfactory bulb (OB) features continued, activity-dependent integration of adult-born neurons, providing a robust model with which to examine mechanisms of plasticity in the adult brain. We previously reported that local OB interneurons secrete the neuropeptide corticotropin-releasing hormone (CRH) in an activity-dependent manner onto adult-born granule neurons and that local CRH signaling promotes expression of synaptic machinery in the bulb. This effect is mediated via activation of the CRH receptor 1 (CRHR1), which is developmentally regulated during adult-born neuron maturation. CRHR1 is a GS-protein-coupled receptor that activates CREB-dependent transcription in the presence of CRH. Therefore, we hypothesized that locally secreted CRH activates CRHR1 to initiate circuit plasticity programs. To identify such programs, we profiled gene expression changes associated with CRHR1 activity in adult-born neurons of the OB. Here, we show that CRHR1 activity influences expression of the brain-specific Homeobox-containing transcription factor POU Class 6 Homeobox 1 (POU6f1). To elucidate the contributions of POU6f1 toward activity-dependent circuit remodeling, we targeted CRHR1+ neurons in male and female mice for cell-type-specific manipulation of POU6f1 expression. Whereas loss of POU6f1 in CRHR1+ neurons resulted in reduced dendritic complexity and decreased synaptic connectivity, overexpression of POU6f1 in CRHR1+ neurons promoted dendritic outgrowth and branching and influenced synaptic function. Together, these findings suggest that the transcriptional program directed by POU6f1 downstream of local CRH signaling in adult-born neurons influences circuit dynamics in response to activity-dependent peptide signaling in the adult brain.SIGNIFICANCE STATEMENT Elucidating mechanisms of plasticity in the adult brain is helpful for devising strategies to understand and treat neurodegeneration. Circuit plasticity in the adult mouse olfactory bulb is exemplified by both continued cell integration and synaptogenesis. We previously reported that these processes are influenced by local neuropeptide signaling in an activity-dependent manner. Here, we show that local corticotropin-releasing hormone (CRH) signaling induces dynamic gene expression changes in CRH receptor expressing adult-born neurons, including altered expression of the transcription factor POU6f1 We further show that POU6f1 is necessary for proper dendrite specification and patterning, as well as synapse development and function in adult-born neurons. Together, these findings reveal a novel mechanism by which peptide signaling modulates adult brain circuit plasticity.

Tau polarizes an aging transcriptional signature to excitatory neurons and glia.

Aging is a major risk factor for Alzheimer's disease (AD), and cell-type vulnerability underlies its characteristic clinical manifestations. We have performed longitudinal, single-cell RNA-sequencing in Drosophila with pan-neuronal expression of human tau, which forms AD neurofibrillary tangle pathology. Whereas tau- and aging-induced gene expression strongly overlap (93%), they differ in the affected cell types. In contrast to the broad impact of aging, tau-triggered changes are strongly polarized to excitatory neurons and glia. Further, tau can either activate or suppress innate immune gene expression signatures in a cell-type-specific manner. Integration of cellular abundance and gene expression pinpoints nuclear factor kappa B signaling in neurons as a marker for cellular vulnerability. We also highlight the conservation of cell-type-specific transcriptional patterns between Drosophila and human postmortem brain tissue. Overall, our results create a resource for dissection of dynamic, age-dependent gene expression changes at cellular resolution in a genetically tractable model of tauopathy.

Co-transmitting neurons in the lateral septal nucleus exhibit features of neurotransmitter switching.

The lateral septal nucleus (LSN) is a highly interconnected region of the central brain whose activity regulates widespread circuitry. As such, the mechanisms that govern neuronal activity within the LSN have far-reaching implications on numerous brain-wide nuclei, circuits, and behaviors. We found that GABAergic neurons within the LSN express markers that mediate the release of acetylcholine (ACh). Moreover, we show that these vGATLSN neurons release both GABA and ACh onto local glutamatergic LSN neurons. Using both short-term and long-term neuronal labeling techniques we observed expression of the cholinergic neuron marker Choline Acetyltransferase (ChAT) in vGATLSN neurons. These findings provide evidence of cholinergic neurotransmission from vGATLSN neurons, and provide an impetus to examine dynamic co-neurotransmission changes as a potential mechanism that contributes to neuronal and circuit-wide plasticity within the LSN.

Activation of basal forebrain-to-lateral habenula circuitry drives reflexive aversion and suppresses feeding behavior.

Environmental cues and internal states such as mood, reward, or aversion directly influence feeding behaviors beyond homeostatic necessity. The hypothalamus has been extensively investigated for its role in homeostatic feeding. However, many of the neural circuits that drive more complex, non-homeostatic feeding that integrate valence and sensory cues (such as taste and smell) remain unknown. Here, we describe a basal forebrain (BF)-to-lateral habenula (LHb) circuit that directly modulates non-homeostatic feeding behavior. Using viral-mediated circuit mapping, we identified a population of glutamatergic neurons within the BF that project to the LHb, which responds to diverse sensory cues, including aversive and food-related odors. Optogenetic activation of BF-to-LHb circuitry drives robust, reflexive-like aversion. Furthermore, activation of this circuitry suppresses the drive to eat in a fasted state. Together, these data reveal a role of basal forebrain glutamatergic neurons in modulating LHb-associated aversion and feeding behaviors by sensing environmental cues.

Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing.

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the morphology of processes and substructures within these neurons. As such, much of what is known about brain function has arisen alongside developments in imaging technologies that allow further insight into how neurons are organized and connected in the brain. Improvements in tissue clearing have allowed for high-resolution imaging of thick brain slices, facilitating morphological reconstruction and analyses of neuronal substructures, such as dendritic arbors and spines. In tandem, advances in image processing software provide methods of quickly analyzing large imaging datasets. This work presents a relatively rapid method of processing, visualizing, and analyzing thick slices of labeled neural tissue at high-resolution using CLARITY tissue clearing, confocal microscopy, and image analysis. This protocol will facilitate efforts toward understanding the connectivity patterns and neuronal morphologies that characterize healthy brains, and the changes in these characteristics that arise in diseased brain states.

Olfactory bulb astrocytes mediate sensory circuit processing through Sox9 in the mouse brain.

The role of transcription factors during astrocyte development and their subsequent effects on neuronal development has been well studied. Less is known about astrocytes contributions towards circuits and behavior in the adult brain. Astrocytes play important roles in synaptic development and modulation, however their contributions towards neuronal sensory function and maintenance of neuronal circuit architecture remain unclear. Here, we show that loss of the transcription factor Sox9 results in both anatomical and functional changes in adult mouse olfactory bulb (OB) astrocytes, affecting sensory processing. Indeed, astrocyte-specific deletion of Sox9 in the OB results in decreased odor detection thresholds and discrimination and it is associated with aberrant neuronal sensory response maps. At functional level, loss of astrocytic Sox9 impairs the electrophysiological properties of mitral and tufted neurons. RNA-sequencing analysis reveals widespread changes in the gene expression profiles of OB astrocytes. In particular, we observe reduced GLT-1 expression and consequential alterations in glutamate transport. Our findings reveal that astrocytes are required for physiological sensory processing and we identify astrocytic Sox9 as an essential transcriptional regulator of mature astrocyte function in the mouse OB.

Oxytocin and Sensory Network Plasticity.

An essential characteristic of nervous systems is their capacity to reshape functional connectivity in response to physiological and environmental cues. Endogenous signals, including neuropeptides, governs nervous system plasticity. Particularly, oxytocin has been recognized for its role in mediating activity-dependent circuit changes. These oxytocin-dependent changes occur at the synaptic level and consequently shape the cellular composition of circuits. Here we discuss recent advances that illustrate how oxytocin functions to reshape neural circuitry in response to environmental changes. Excitingly, recent findings pave the way for promising therapeutic applications of oxytocin to treat neurodevelopmental and neuropsychiatric diseases.

Target specific functions of EPL interneurons in olfactory circuits.

Inhibitory interneurons are integral to sensory processing, yet revealing their cell type-specific roles in sensory circuits remains an ongoing focus. To Investigate the mouse olfactory system, we selectively remove GABAergic transmission from a subset of olfactory bulb interneurons, EPL interneurons (EPL-INs), and assay odor responses from their downstream synaptic partners - tufted cells and mitral cells. Using a combination of in vivo electrophysiological and imaging analyses, we find that inactivating this single node of inhibition leads to differential effects in magnitude, reliability, tuning width, and temporal dynamics between the two principal neurons. Furthermore, tufted and not mitral cell responses to odor mixtures become more linearly predictable without EPL-IN inhibition. Our data suggest that olfactory bulb interneurons, through exerting distinct inhibitory functions onto their different synaptic partners, play a significant role in the processing of odor information.

Single-Cell RNA-Seq of Mouse Olfactory Bulb Reveals Cellular Heterogeneity and Activity-Dependent Molecular Census of Adult-Born Neurons.

Cellular heterogeneity within the mammalian brain poses a challenge toward understanding its complex functions. Within the olfactory bulb, odor information is processed by subtypes of inhibitory interneurons whose heterogeneity and functionality are influenced by ongoing adult neurogenesis. To investigate this cellular heterogeneity and better understand adult-born neuron development, we utilized single-cell RNA sequencing and computational modeling to reveal diverse and transcriptionally distinct neuronal and nonneuronal cell types. We also analyzed molecular changes during adult-born interneuron maturation and uncovered developmental programs within their gene expression profiles. Finally, we identified that distinct neuronal subtypes are differentially affected by sensory experience. Together, these data provide a transcriptome-based foundation for investigating subtype-specific neuronal function in the olfactory bulb (OB), charting the molecular profiles that arise during the maturation and integration of adult-born neurons and how they dynamically change in an activity-dependent manner.

Olfactory Cued Learning Paradigm.

Sensory stimulation leads to structural changes within the CNS (Central Nervous System), thus providing the fundamental mechanism for learning and memory. The olfactory circuit offers a unique model for studying experience-dependent plasticity, partly due to a continuous supply of integrating adult born neurons. Our lab has recently implemented an olfactory cued learning paradigm in which specific odor pairs are coupled to either a reward or punishment to study downstream circuit changes. The following protocol outlines the basic set up for our learning paradigm. Here, we describe the equipment setup, programming of software, and method of behavioral training.