Biology subjects

McCreedy, D. A.

Publications and source records attributed to McCreedy, D. A..

5 recordsLinked to original sources

bioRxiv2026

A direct SCN-to-DMH output pathway organizes circadian behavioral timing

Circadian rhythms in behavior depend on the suprachiasmatic nucleus (SCN), but how SCN timekeeping is transmitted to downstream circuits that organize daily behavioral rhythms remains poorly defined. The dorsomedial hypothalamus (DMH) has been implicated in circadian behavioral output, but lesion studies cannot determine whether the DMH contributes through local molecular timekeeping or through intact neurons that relay SCN-derived timing signals. Here, we combined DMH neuronal ablation, local molecular clock disruption, retrograde and intersectional tracing, single-cell RNA sequencing, and intersectional optogenetics to define and test a direct SCN-to-DMH output pathway. DMH neuronal ablation disrupted locomotor activity rhythms, whereas DMH Cry1/2 disruption did not, indicating that intact DMH neurons, but not local molecular timekeeping, are required for locomotor rhythmicity. Retrograde tracing identified a sparse population of DMH-projecting SCN neurons concentrated in the dorsal SCN. These neurons showed minimal overlap with canonical AVP- and VIP-expressing SCN populations and were most strongly represented in a Prokr2/Vipr2-expressing transcriptional cluster. Repeated activation of DMH-projecting SCN neurons entrained locomotor rhythms, produced persistent phase shifts after stimulation ended, and compressed the temporal distribution of activity during entrainment. Together, these findings identify sparse, molecularly distinct DMH-projecting SCN neurons capable of organizing circadian locomotor timing.

neuroscience↗

bioRxiv2025

Sex-dependent effects of peptidylarginine deiminases on neutrophil function and long-term outcomes after spinal cord injury

Traumatic spinal cord injury (SCI) initiates an influx of peripheral immune cells to the spinal cord parenchyma that compound tissue damage and restrict functional recovery. Neutrophils infiltrate the spinal cord within the first day after injury, releasing extracellular traps (NETs) comprised of decondensed DNA, modified histones, and granule enzymes, that can worsen tissue damage. Peptidylarginine demininases (PADs), particularly PAD4, have been indicated as mediators of NET formation by facilitating the decondensation of nuclear chromatin via histone citrullination. Though PADs have been shown to be regulated by sex hormones, sex-differences in PAD regulation of neutrophil function in the context of CNS injury have yet to be explored. In this work, we investigated the role of PADs in recovery after SCI using Cl-amidine, a pan-PAD inhibitor. Strikingly, Cl-amidine treated mice exhibited sex-dependent changes to motor function, body weight, and white matter sparing after SCI. Acutely, Cl-amidine treated mice had reduced NET accumulation in the blood and decreased spinal cord neutrophil granularity. Analysis of publicly available scRNA-seq data revealed that female bone marrow neutrophils exhibited elevated Padi4 expression relative to their male counterparts. We then utilized Padi4 knockout (Padi4-/-) mice to assess the role of PAD4 in long-term recovery of male and female mice after SCI. While we observed no changes in motor recovery, a sex-dependent effect on tissue sparing was observed with Padi4 deficiency. These data are the first description of sex differences in PAD-mediated neutrophil function after SCI and highlight the importance of inclusion of both sexes in pre-clinical research.

neuroscience↗

bioRxiv2025

Functional synaptic connectivity of engrafted spinal cord neurons with locomotor circuitry in the injured spinal cord

Spinal cord injury (SCI) results in significant neurological deficits, with no currently available curative therapies. Neural progenitor cell (NPC) transplantation has emerged as a promising approach for neural repair, as graft-derived neurons (GDNs) can integrate into the host spinal cord and support axon regeneration. However, the mechanisms underlying functional recovery remain poorly understood. In this study, we investigate the synaptic integration of NPC-derived neurons into locomotor circuits, the projection patterns of distinct neuronal subtypes, and their potential to modulate motor circuit activity. Using transsynaptic tracing in a mouse thoracic contusion SCI model, we found that NPC-derived neurons form synaptic connections with host locomotor circuits, albeit at low frequencies. Furthermore, we mapped the axon projections of V0C and V2a interneurons, revealing distinct termination patterns within host spinal cord laminae. To assess functional integration, we employed chemogenetic activation of GDNs, which induced muscle activity in a subset of transplanted animals. However, NPC transplantation alone did not significantly improve locomotor recovery, highlighting a key challenge in the field. Our findings suggest that while GDNs can integrate into host circuits and modulate motor activity, synaptic connectivity remains a limiting factor in functional recovery. Future studies should focus on enhancing graft-host connectivity and optimizing transplantation strategies to maximize therapeutic benefits for SCI.

neuroscience↗

bioRxiv2025

Rapid and efficient optical tissue clearing for volumetric imaging of the intact and injured spinal cord in mice

Tissue clearing and 3D imaging have emerged as powerful techniques to assess the cellular and tissue-level architecture of the spinal cord. With the rapidly increasing variety and complexity of optical tissue clearing techniques, there is a critical need for optimization and streamlining of tissue-specific protocols, particularly when dealing with injury or disease states. We evaluated and combined multiple organic solvent-based techniques to develop sciDISCO: a spinal cord injury-optimized DISCO tissue clearing protocol. sciDISCO allows for the robust clearing, labeling, and 3D imaging of the intact spinal cord, as well as clearing around and through the lesion site formed after contusive spinal cord injury. In addition, we have identified alternatives for hazardous chemicals commonly used in organic solvent-based clearing including dichloromethane and dibenzyl ether. In this study, we demonstrate the compatibility of sciDISCO with multiple different labeling techniques to provide robust analysis of unique neuronal populations and morphologies in addition to cellular and tissue-level changes occurring following spinal cord injury.

neuroscience↗

bioRxiv2021

Passive clearing and 3D lightsheet imaging of the intact and injured spinal cord in mice

The spinal cord contains a diverse array of sensory and motor circuits that are essential for normal function. Spinal cord injury (SCI) permanently disrupts neural circuits through initial mechanical damage, as well as a cascade of secondary injury events that further expand the spinal cord lesion, resulting in permanent paralysis. Tissue clearing and 3D imaging have recently emerged as promising techniques to improve our understanding of the complex neural circuitry of the spinal cord and the changes that result from damage due to SCI. However, the application of this technology for studying the intact and injured spinal cord remains limited. Here we optimized the passive CLARITY technique (PACT) to obtain gentle and efficient clearing of the murine spinal cord without the need for specialized equipment. We demonstrate that PACT clearing enables 3D imaging of multiple fluorescent labels in the spinal cord to assess molecularly defined neuronal populations, acute inflammation, long-term tissue damage, and cell transplantation. Collectively, these procedures provide a framework for expanding the utility of tissue clearing to enhance the study of spinal cord neural circuits, as well as cellular- and tissue-level changes that occur following SCI.

neuroscience↗