Biology subjects

DeNardo, L. A.

Publications and source records attributed to DeNardo, L. A..

13 recordsLinked to original sources

bioRxiv2025

Early adversity potentiates prefrontal - amygdala communication through CRH+ glutamatergic neurons

Early life adversity (ELA) has lasting impacts on emotion circuit function1. Exposure to early maltreatment, neglect, or household instability is associated with enduring alterations in threat evaluation and response, hallmarks of mood and anxiety disorders2-4. These alterations often emerge during adolescence and are characterized by excessive avoidance that limits engagement in rewarding experiences5,6. However, the neurodevelopmental mechanisms linking ELA to increased threat responses remain unclear. We identified a stress-sensitive medial prefrontal cortex (mPFC)-basolateral amygdala pathway that co-expresses corticotropin-releasing hormone (CRH) and glutamate. We find that ELA strengthens this pathway and increases threat avoidance behavior during adolescence. Optogenetic inhibition and developmental CRH knockdown in this pathway both rescue ELA-induced elevations in adolescent avoidance, whereas broadly reducing mPFC CRH expression in typically reared mice mimics ELA phenotypes. The same manipulations produced no behavioral effects in adults. Together, our findings indicate that cell type-specific CRH signaling regulates developmental circuit plasticity, biasing emotional circuit function and potentially contributing to psychiatric vulnerability.

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bioRxiv2025

Directed cell-type recruitment during consolidation of a remote memory

Memories are consolidated into a distributed neocortical network for long-term storage. Long-term memory retrieval relies on cells that are active during learning and undergo necessary plasticity. However, remote memory retrieval activates a broader circuit, with learning-activated cells representing only a small subset. What are the rules and cell-types governing memory trace reorganization? We identified a class of prefrontal projection neurons that are gradually recruited to a memory trace through synaptic activity of learning-activated cells. This population, which projects to the temporal association area (TEa), progressively strengthens its encoding of memory-induced behaviors, mirroring increases in TEa activity. Notably, the prefrontal-TEa pathway is required for remote but not recent memory retrieval. Our findings reveal a cell type-specific mechanism underlying memory trace reorganization during consolidation.

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bioRxiv2025

A cell type-specific mechanism driving the rapid antidepressant effects of transcranial magnetic stimulation

Repetitive transcranial magnetic stimulation (rTMS) is an emerging treatment for brain disorders, but its therapeutic mechanism is unknown. We developed a novel mouse model of rTMS with superior clinical face validity and investigated the neural mechanism by which accelerated intermittent theta burst stimulation (aiTBS) - the first rapid-acting rTMS antidepressant protocol - reversed chronic stress-induced behavioral deficits. Using fiber photometry, we showed that aiTBS drives distinct patterns of neural activity in intratelencephalic (IT) and pyramidal tract (PT) projecting neurons in dorsomedial prefrontal cortex (dmPFC). However, only IT neurons exhibited persistently increased activity during both aiTBS and subsequent depression-related behaviors. Similarly, aiTBS reversed stress-related loss of dendritic spines on IT, but not PT neurons, further demonstrating cell type-specific effects of stimulation. Finally, chemogenetic inhibition of dmPFC IT neurons during rTMS blocked the antidepressant-like behavioral effects of aiTBS. Thus, we demonstrate a prefrontal mechanism linking rapid aiTBS-driven therapeutic effects to cell type-specific circuit plasticity.

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bioRxiv2025

Microglia regulate nucleus accumbens synaptic development and circuit function underlying threat avoidance behaviors

While CNS microglia have well-established roles in synapse pruning during neurodevelopment, only a few studies have identified roles for microglia in synapse formation. These studies focused on the cortex and primary sensory circuits during restricted developmental time periods, leaving substantial gaps in our understanding of the early developmental functions of microglia. Here we investigated how the absence of microglia impacts synaptic development in the nucleus accumbens (NAc), a region critical for emotional regulation and motivated behaviors and where dysfunction is implicated in psychiatric disorders that arise early in life. Using a genetically modified mouse that lacks microglia (Csf1r {Delta}FIRE/{Delta}FIRE), we found blunted excitatory synapse formation in the NAc. This effect was most prominent during the second and third postnatal weeks, when we previously found microglia to be overproduced, and was accompanied by an increase in presynaptic release probability and alterations in postsynaptic kinetics. Tissue-level NAc proteomics confirmed that microglial absence impacted numerous proteins involved in synapse structure, trans-synaptic signaling, and pre-synaptic function. However, microglial absence did not perturb levels of astrocyte-derived cues and adhesive proteins that promote synaptogenesis, suggesting that reduced synapse number may be caused by absence of a microglial-derived synaptogenic cue. Although observed electrophysiological synaptic changes were largely normalized by adulthood, we identified lasting effects of microglial absence on threat avoidance behavior, and these behavioral effects were directly associated with alterations of NAc neuronal activity. Together, these results indicate a critical role for microglia in regulating the synaptic landscape of the developing NAc and in establishing functional circuits underlying adult behavioral repertoires.

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bioRxiv2024

Sex-and Stress-Dependent Plasticity of a Corticotropin Releasing Hormone / GABA Projection from the Basolateral Amygdala to Nucleus Accumbens that Mediates Reward Behaviors

BackgroundMotivated behaviors are executed by refined brain circuits. Early-life adversity (ELA) is a risk for human affective disorders involving dysregulated reward behaviors. In mice, ELA causes anhedonia-like behaviors in males and augmented reward motivation in females, indicating sex-dependent disruption of reward circuit operations. We recently identified a corticotropin-releasing hormone (CRH) expressing GABAergic projection from basolateral amygdala (BLA) to nucleus accumbens (NAc) that governs reward-seeking deficits in adult ELA males--but not females. MethodsTo probe the sex-specific role of this projection in reward behaviors, adult male and female CRH-Cre mice raised in control or ELA conditions received excitatory or inhibitory Cre-dependent DREADDs in BLA, and then clozapine N-oxide or vehicle to NAc medial shell during reward behaviors. We determined the cell identity of the projection using immunostaining and electrophysiology. Using tissue clearing, light sheet fluorescence microscopy and deep learning pipelines, we mapped brain-wide BLA CRH+ axonal projections to uncover sex differences in innervation. ResultsChemogenetic manipulations in male mice demonstrated inhibitory effects of the CRH+ BLA-NAc projection on reward behaviors, whereas neither excitation nor inhibition influenced female behaviors. Molecular and electrophysiological cell-identities of the projection did not vary by sex. By contrast, comprehensive whole-brain mapping uncovered significant differences in NAc innervation patterns that were both sex and ELA-dependent, as well as selective changes of innervation of other brain regions. ConclusionsThe CRH/GABA BLA-NAc projection that influences reward behaviors in males differs structurally and functionally in females, uncovering potential mechanisms for the profound sex-specific impacts of ELA on reward behaviors.

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bioRxiv2024

Transformations in prefrontal ensemble activity underlying rapid threat avoidance learning

The capacity to learn cues that predict aversive outcomes, and understand how to avoid those outcomes, is critical for adaptive behavior. Naturalistic avoidance often means accessing a safe location, but whether a location is safe depends on the nature of the impending threat. These relationships must be rapidly learned if animals are to survive. The prelimbic subregion (PL) of the medial prefrontal cortex (mPFC) integrates learned associations to influence these threat avoidance strategies. Prior work has focused on the role of PL activity in avoidance behaviors that are fully established, leaving the prefrontal mechanisms that drive rapid avoidance learning poorly understood. To determine when and how these learning-related changes emerge, we recorded PL neural activity using miniscope calcium imaging as mice rapidly learned to avoid a threatening cue by accessing a safe location. Over the course of learning, we observed enhanced modulation of PL activity representing intersections of a threatening cue with safe or risky locations and movements between them. We observed rapid changes in PL population dynamics that preceded changes observable in the encoding of individual neurons. Successful avoidance could be predicted from cue-related population dynamics during early learning. Population dynamics during specific epochs of the conditioned tone period correlated with the modeled learning rates of individual animals. In contrast, changes in single-neuron encoding occurred later, once an avoidance strategy had stabilized. Together, our findings reveal the sequence of PL changes that characterize rapid threat avoidance learning.

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bioRxiv2024

Prefrontal dopamine circuits are required for active avoidance learning but not fear learning

To survive, animals must rapidly learn to avoid predictable threats. Such learning depends on detecting reliable cue-outcome relationships that efficiently drive behavioral adaptations. The medial prefrontal cortex (mPFC) integrates learned information about the environment to guide adaptive behaviors1-7 and is critical for threat avoidance8-15. However, as most studies have focused on well-learned threat avoidance strategies, the specific inputs that signal avoidability and drive rapid avoidance learning remain poorly understood. Dopamine (DA) inputs from the ventral tegmental area (VTA) potently modulate prefrontal function and are preferentially engaged by aversive stimuli16-21. Previous studies implicated VTA and prefrontal DA in aversive learning22-25, but their findings have been constrained by limited spatiotemporal resolution. Here, we used high-resolution tools to dissect the role of the VTA-mPFC DA circuit in rapid avoidance learning. Optogenetic suppression of VTA DA terminals in mPFC selectively impaired learning of a cued avoidance response, without affecting cue-shock association learning, reactive escape behaviors, or expression of previously learned avoidance. Using a fluorescent DA sensor, we observe rapid, event-locked DA activity that emerged transiently during the initiation of learning. Increased DA encoded aversive outcomes and their predictive cues, while decreased DA encoded their omission and predicted how quickly mice learned to avoid. In yoked mice lacking control over shock omission, these dynamics were largely absent. Together, these findings demonstrate that the VTA-mPFC DA circuit is necessary for rapid acquisition of proactive avoidance behaviors and reveal transient event-related DA signals that underlie this form of learning.

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bioRxiv2024

A developmental brain-wide screen identifies retrosplenial cortex as a key player in the emergence of persistent memory

Memories formed early in life are short-lived while those formed later persist. Recent work revealed that infant memories are stored in a latent state. But why they fail to be retrieved is poorly understood. Here we investigated brain-wide circuit mechanisms underlying infantile amnesia. We performed a screen that combined contextual fear conditioning, activity-dependent neuronal tagging at different postnatal ages, tissue clearing and light sheet microscopy. We observed striking developmental changes in regional activity patterns between infant, juvenile, and adult mice, including changes in the retrosplenial cortex (RSP) that aligned with the emergence of persistent memory. We then performed a series of targeted investigations of RSP structure and function across development. Chronic chemogenetic reactivation of tagged RSP ensembles during the week after learning enhanced memory in adults and juveniles, but not in infants. However, after 33 days, reactivating infant-tagged RSP ensembles recovered forgotten memories. Changes in the developmental functions of RSP memory ensembles were accompanied by changes in dendritic spine density and the likelihood that those ensembles could be reactivated by contextual cues. These studies show that RSP ensembles store latent infant memories, reveal the time course of RSP functional maturation, and suggest that immature RSP functional networks contribute to infantile amnesia.

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bioRxiv2023

Developmentally distinct architectures in top-down circuits

The medial prefrontal cortex (mPFC) plays a key role in learning, mood and decision making, including in how individuals respond to threats1-6. mPFC undergoes a uniquely protracted development, with changes in synapse density, cortical thickness, long-range connectivity, and neuronal encoding properties continuing into early adulthood7-21. Models suggest that before adulthood, the slow-developing mPFC cannot adequately regulate activity in faster-developing subcortical centers22,23. They propose that during development, the enhanced influence of subcortical systems underlies distinctive behavioural strategies of juveniles and adolescents and that increasing mPFC control over subcortical structures eventually allows adult behaviours to emerge. Yet it has remained unclear how a progressive strengthening of top-down control can lead to nonlinear changes in behaviour as individuals mature24,25. To address this discrepancy, here we monitored and manipulated activity in the developing brain as animals responded to threats, establishing direct causal links between frontolimbic circuit activity and the behavioural strategies of juvenile, adolescent and adult mice. Rather than a linear strengthening of mPFC synaptic connectivity progressively regulating behaviour, we uncovered multiple developmental switches in the behavioural roles of mPFC circuits targeting the basolateral amygdala (BLA) and nucleus accumbens (NAc). We show these changes are accompanied by axonal pruning coinciding with functional strengthening of synaptic connectivity in the mPFC-BLA and mPFC-NAc pathways, which mature at different rates. Our results reveal how developing mPFC circuits pass through distinct architectures that may make them optimally adapted to the demands of age-specific challenges.

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bioRxiv2022

Mapping whole-brain projections of anatomically defined prefrontal neurons using combined 3D convolution networks

To understand how the brain produces behavior, we must elucidate the relationships between neuronal connectivity and function. The medial prefrontal cortex (mPFC) is critical for complex functions including decision-making and mood. mPFC projection neurons collateralize extensively, but the relationships between mPFC neuronal activity and brain-wide connectivity are poorly understood. We performed whole-brain connectivity mapping and fiber photometry to better understand the mPFC circuits that control threat avoidance. Using tissue clearing and light sheet fluorescence microscopy we mapped the brain-wide axon collaterals of populations of mPFC neurons that project to nucleus accumbens (NAc), ventral tegmental area (VTA), or contralateral mPFC (cmPFC) in mice. We present DeepTraCE, for quantifying bulk-labeled axonal projections in images of cleared tissue, and DeepCOUNT, for quantifying cell bodies. Anatomical maps produced with DeepTraCE aligned with known axonal projection patterns and revealed class-specific topographic projections within regions. During threat avoidance, cmPFC and NAc-projectors encoded conditioned stimuli, but only when action was required to avoid threats. mPFC-VTA neurons encoded learned but not innate avoidance behaviors. Together our results present new and optimized approaches for quantitative whole-brain analysis and indicate that anatomically-defined classes of mPFC neurons have specialized roles in threat avoidance.

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bioRxiv2021

BehaviorDEPOT: a tool for automated behavior classification and analysis in rodents

Quantitative descriptions of animal behavior are essential to understand the underlying neural substrates. Many behavioral analyses are performed by hand or with expensive and inflexible commercial software that often fail on animals with attached head implants, such as those used for in vivo optogenetics and calcium imaging. With the development of machine learning algorithms that can estimate animal positions across time and space, it is becoming easier for users with no prior coding experience to perform automated animal tracking in behavioral video recordings. Yet classifying discrete behaviors based on positional tracking data remains a significant challenge. To achieve this, we must start with reliable ground truth definitions of behavior, a process that is hindered by unreliable human annotations. To overcome these barriers, we developed BehaviorDEPOT (DEcoding behavior based on POsitional Tracking), a MATLAB-based application comprising six independent modules and a graphical user interface. In the Analysis Module we provide hard-coded classifiers for freezing and rearing. Optionally applied spatiotemporal filters allow users to analyze behaviors in varied experimental designs (e.g. cued tasks or optogenetic manipulations). Even inexperienced users can generate organized behavioral data arrays that can be seamlessly aligned with neurophysiological recordings for detailed analyses of the neural substrates. Four additional modules create an easy-to-use pipeline for establishing reliable ground-truth definitions of behaviors as well as custom behavioral classifiers. Finally, our Experiment Module runs fear conditioning experiments using an Arduino-based design that interfaces with commercialhardware and significantly reduces associated costs. We demonstrate the utility and flexibility of BehaviorDEPOT in widely used behavioral assays including fear conditioning, avoidance, and decision-making tasks. We also demonstrate the robustness of the BehaviorDEPOT freezing classifier across multiple camera types and in mice and rats wearing optogenetic patch cables and head-mounted Miniscopes. BehaviorDEPOT provides a simple, flexible, automated pipeline to move from pose tracking to reliably quantifying a wide variety of task-relevant behaviors.

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bioRxiv2018

Temporal Evolution of Cortical Ensembles Promoting Remote Memory Retrieval

Studies of amnesic patients and animal models support a systems consolidation model, which posits that explicit memories formed in hippocampus are transferred to cortex over time1-6. Prelimbic cortex (PL), a subregion of the medial prefrontal cortex, is required for the expression of learned fear memories from hours after learning until weeks later7-12. While some studies suggested that prefrontal cortical neurons active during learning are required for memory retrieval13-15, others provided evidence for ongoing cortical circuit reorganization during memory consolidation10,16,17. It has been difficult to causally relate the activity of cortical neurons during learning or recent memory retrieval to their function in remote memory, in part due to a lack of tools18. Here we show that a new version of targeted recombination in active populations, TRAP2, has enhanced efficiency over the past version, providing brain-wide access to neurons activated by a particular experience. Using TRAP2, we accessed PL neurons activated during fear conditioning or 1-, 7-, or 14-day memory retrieval, and assessed their contributions to 28-day remote memory. We found that PL neurons TRAPed at later retrieval times were more likely to be reactivated during remote memory retrieval, and more effectively promoted remote memory retrieval. Furthermore, reducing PL activity during learning blunted the ability of TRAPed PL neurons to promote remote memory retrieval. Finally, a series of whole-brain analyses identified a set of cortical regions that were densely innervated by memory-TRAPed PL neurons and preferentially activated by PL neurons TRAPed during 14-day retrieval, and whose activity co-varied with PL and correlated with memory specificity. These findings support a model in which PL ensembles underlying remote memory undergo dynamic changes during the first two weeks after learning, which manifest as increased functional recruitment of cortical targets.

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bioRxiv2017

Amyloid accumulation drives proteome-wide alterations in mouse models of Alzheimers disease like pathology

Amyloid beta (A{beta}) peptides impair multiple cellular pathways in the brain and play a causative role in Alzheimers disease (AD) pathology, but how the brain proteome is remodeled during this process is unknown. To identify new protein networks associated with AD-like pathology, we performed global quantitative proteomic analysis in three mouse models at pre- and post-symptomatic ages. Our analysis revealed a robust and consistent increase in Apolipoprotein E (ApoE) levels in nearly all transgenic brain regions with increased A{beta} levels. Taken together with prior findings on ApoE driving A{beta} accumulation, this analysis points to a pathological dysregulation of the ApoE-A{beta} axis. We also found dysregulation of protein networks involved in excitatory synaptic transmission consistent with AD pathophysiology. Targeted analysis of the AMPA receptor complex revealed a specific loss of TARP{gamma}-2, a key AMPA receptor trafficking protein. Expression of TARP{gamma}-2 in vivo in hAPP transgenic mice led to a restoration of AMPA currents. This database of proteome alterations represents a unique resource for the identification of protein alterations responsible for AD.\n\nHighlightsO_LIProteomic analysis of mouse brains with AD-like pathology reveals stark remodeling\nC_LIO_LIProteomic evidence points to a dysregulation of ApoE levels associated with A{beta} clearance rather than production\nC_LIO_LICo-expression analysis found distinctly impaired synapse and mitochondria modules\nC_LIO_LIIn-depth analyses of AMPAR complex points to loss of TARP{gamma}-2, which may compromise synapses in AD\nC_LI\n\neTOC BlurbProteome-wide profiling of brain tissue from three mouse models of AD-like pathology reveals A{beta}, brain region, and age dependent alterations of protein levels. This resource provides a new global protein expression atlas for the Alzheimers disease research community.

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