Parkinson's disease (PD) develops over decades through spatiotemporal stages that ascend from the brainstem to the forebrain. The mechanism behind this caudo-rostral neurodegeneration remains largely undefined. In unraveling this phenomenon, we recently developed a lipopolysaccharide (LPS)-elicited chronic neuroinflammatory mouse model that displays sequential losses of neurons in brainstem, substantia nigra, hippocampus and cortex. In this study, we aimed to investigate the mechanisms of caudo-rostral neurodegeneration and focused our efforts on the earliest neurodegeneration of vulnerable noradrenergic locus coeruleus (NE-LC) neurons in the brainstem. We found that compared with neurons in other brain regions, NE-LC neurons in untreated mice displayed high levels of mitochondrial oxidative stress that was severely exacerbated in the presence of LPS-elicited chronic neuroinflammation. In agreement, NE-LC neurons in LPS-treated mice displayed early reduction of complex IV expression and mitochondrial swelling and loss of cristae. Mechanistically, the activation of the superoxide-generating enzyme NADPH oxidase (NOX2) on NE-LC neurons was essential for their heightened vulnerability during chronic neuroinflammation. LPS induced early and high expressions of NOX2 in NE-LC neurons. Genetic or pharmacological inactivation of NOX2 markedly reduced mitochondrial oxidative stress and dysfunction in LPS-treated mice. Furthermore, inhibition of NOX2 significantly ameliorated LPS-induced NE-LC neurodegeneration. More importantly, post-treatment with NOX2 inhibitor diphenyleneiodonium when NE-LC neurodegeneration had already begun, still showed high efficacy in protecting NE-LC neurons from degeneration in LPS-treated mice. This study strongly supports that chronic neuroinflammation and NOX2 expression among vulnerable neuronal populations contribute to caudo-rostral degeneration in PD.

The default mode network (DMN) of the brain is functionally associated with a wide range of behaviors. In this study, we used functional magnetic resonance imaging (fMRI), positron emission tomography (PET), and spectral fiber photometry to investigate the selective neuromodulatory effect of norepinephrine (NE)-releasing noradrenergic neurons in the locus coeruleus (LC) on the mouse DMN. Chemogenetic-induced tonic LC activity decreased cerebral blood volume (CBV) and glucose uptake and increased synchronous low-frequency fMRI activity within the frontal cortices of the DMN. Fiber photometry results corroborated these findings, showing that LC-NE activation induced NE release, enhanced calcium-weighted neuronal spiking, and reduced CBV in the anterior cingulate cortex. These data suggest that LC-NE alters conventional coupling between neuronal activity and CBV in the frontal DMN. We also demonstrated that chemogenetic activation of LC-NE neurons strengthened functional connectivity within the frontal DMN, and this effect was causally mediated by reduced modulatory inputs from retrosplenial and hippocampal regions to the association cortices of the DMN.

Noradrenergic signaling plays a well-established role in promoting the stress response. Here we identify a subpopulation of noradrenergic neurons, defined by developmental expression of Hoxb1, that has a unique role in modulating stress-related behavior. Using an intersectional chemogenetic strategy, in combination with behavioral and physiological analyses, we show that activation of Hoxb1-noradrenergic (Hoxb1-NE) neurons decreases anxiety-like behavior and promotes an active coping strategy in response to acute stressors. In addition, we use cerebral blood volume-weighted functional magnetic resonance imaging to show that chemoactivation of Hoxb1-NE neurons results in reduced activity in stress-related brain regions, including the bed nucleus of the stria terminalis, amygdala, and locus coeruleus. Thus, the actions of Hoxb1-NE neurons are distinct from the well-documented functions of the locus coeruleus in promoting the stress response, demonstrating that the noradrenergic system contains multiple functionally distinct subpopulations.

Alterations in genes that regulate brain size may contribute to both microcephaly and brain tumor formation. Here, we report that Aspm, a gene that is mutated in familial microcephaly, regulates postnatal neurogenesis in the cerebellum and supports the growth of medulloblastoma, the most common malignant pediatric brain tumor. Cerebellar granule neuron progenitors (CGNPs) express Aspm when maintained in a proliferative state by sonic hedgehog (Shh) signaling, and Aspm is expressed in Shh-driven medulloblastoma in mice. Genetic deletion of Aspm reduces cerebellar growth, while paradoxically increasing the mitotic rate of CGNPs. Aspm-deficient CGNPs show impaired mitotic progression, altered patterns of division orientation and differentiation, and increased DNA damage, which causes progenitor attrition through apoptosis. Deletion of Aspm in mice with Smo-induced medulloblastoma reduces tumor growth and increases DNA damage. Co-deletion of Aspm and either of the apoptosis regulators Bax or Trp53 (also known as p53) rescues the survival of neural progenitors and reduces the growth restriction imposed by Aspm deletion. Our data show that Aspm functions to regulate mitosis and to mitigate DNA damage during CGNP cell division, causes microcephaly through progenitor apoptosis when mutated, and sustains tumor growth in medulloblastoma.

Superparamagnetic iron-oxide nanoparticles are robust contrast agents for magnetic resonance imaging (MRI) used for sensitive structural and functional mapping of the cerebral blood volume (CBV) when administered intravenously. To date, many CBV-MRI studies are conducted with Feraheme, manufactured for the clinical treatment of iron-deficiency. Unfortunately, Feraheme is currently not available outside the United States due to commercial and regulatory constraints, making CBV-MRI methods either inaccessible or very costly to achieve. To address this barrier, we developed a simple, one-pot recipe to synthesize Carboxymethyl-dextran coated Iron Oxide Nanoparticles, namely, "CION", suitable for preclinical CBV-MRI applications. Here we disseminate a step-by-step instruction of our one-pot synthesis protocol, which allows CION to be produced in laboratories with minimal cost. We also characterized different CION-conjugations by manipulating polymer to metal stoichiometric ratio in terms of their size, surface chemistry, and chemical composition, and shifts in MR relaxivity and pharmacokinetics. We performed several proof-of-concept experiments in vivo, demonstrating the utility of CION for functional and structural MRI applications, including hypercapnic CO2 challenge, visual stimulation, targeted optogenetic stimulation, and microangiography. We also present evidence that CION can serve as a cross-modality research platform by showing concurrent in vivo optical and MRI measurement of CBV using fluorescent-labeled CION. The simplicity and cost-effectiveness of our one-pot synthesis method should allow researchers to reproduce CION and tailor the relaxivity and pharmacokinetics according to their imaging needs. It is our hope that this work makes CBV-MRI more openly available and affordable for a variety of research applications.

Cortical spreading depolarization (CSD) is known to exacerbate ischemic damage, as the number of CSDs correlates with the final infarct volumes and suppressing CSDs improves functional outcomes. To investigate the role of CSD in ischemic damage, we developed a novel rat model of photothrombotic ischemia using a miniature implantable optic fiber that allows lesion induction inside the magnetic resonance imaging (MRI) scanner. We were able to precisely control the location and the size of the ischemic lesion, and continuously monitor dynamic perfusion and diffusion MRI signal changes at high temporal resolution before, during and after the onset of focal ischemia. Our model showed that apparent diffusion coefficient (ADC) and cerebral blood flow (CBF) in the ischemic core dropped immediately after lesion onset by 20±6 and 41±23%, respectively, and continually declined over the next 5h. Meanwhile, CSDs were observed in all animals (n=36) and displayed either a transient decrease of ADC by 17±3% or an increase of CBF by 104±15%. All CSDs were initiated from the rim of the ischemic core, propagated outward, and confined to the ipsilesional cortex. Additionally, we demonstrated that by controlling the size of perfusion-diffusion mismatch (which approximates the penumbra) in our model, the number of CSDs correlated with the mismatch area rather than the final infarct volume. This study introduces a novel platform to study CSDs in real-time with high reproducibility using MRI.

Microglia and astroglia play critical roles in the development, function, and survival of neurons in the CNS. However, under inflammatory conditions the role of astrogliosis in the inflammatory process and its effects on neurons remains unclear. Here, we used several types of cell cultures treated with the bacterial inflammogen LPS to address these questions. We found that the presence of astroglia reduced inflammation-driven neurotoxicity, suggesting that astrogliosis is principally neuroprotective. Neutralization of supernatant glial cell line-derived neurotrophic factor (GDNF) released from astroglia significantly reduced this neuroprotective effect during inflammation. To determine the immunological role of astroglia, we optimized a highly-enriched astroglial culture protocol and demonstrated that LPS failed to induce the synthesis and release of TNF-α and iNOS/NO. Instead we found significant enhancement of TNF-α and iNOS expression in highly-enriched astroglial cultures required the presence of 0.5-1% microglia, respectively. Thus suggesting that microglial-astroglial interactions are required for LPS to induce the expression of pro-inflammatory factors and GDNF from astroglia. Specifically, we found that microglia-derived TNF-α plays a pivotal role as a paracrine signal to regulate the neuroprotective functions of astrogliosis. Taken together, these findings suggest that astroglia may not possess the ability to directly recognize the innate immune stimuli LPS, but rather depend on crosstalk with microglia to elicit release of neurotrophic factors as a counterbalance to support neuronal survival from the collateral damage generated by activated microglia during neuroinflammation.

Fiber photometry is an emerging technique for recording fluorescent sensor activity in the brain. However, significant hemoglobin absorption artifacts in fiber photometry data may be misinterpreted as sensor activity changes. Because hemoglobin exists widely in the brain, and its concentration varies temporally, such artifacts could impede the accuracy of photometry recordings. Here we present use of spectral photometry and computational methods to quantify photon absorption effects by using activity-independent fluorescence signals, which can be used to derive oxy- and deoxy-hemoglobin concentration changes. Although these changes are often temporally delayed compared with the fast-responding fluorescence spikes, we found that erroneous interpretation may occur when examining pharmacology-induced sustained changes and that sometimes hemoglobin absorption could flip the GCaMP signal polarity. We provide hemoglobin-based correction methods to restore fluorescence signals and compare our results with other commonly used approaches. We also demonstrated the utility of spectral fiber photometry for delineating regional differences in hemodynamic response functions.