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Diet is inextricably linked to human health and biological functionality. Reduced cognitive function among other health issues has been correlated with a western diet (WD) in mouse models, indicating that increases in neurodegeneration could be fueled in part by a poor diet. In this study, we use matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) to spatially map the lipidomic profiles of male and female mice that were fed a high-fat, high-sucrose WD for a period of 7 weeks. Our findings concluded that the cortex and corpus callosum showed significant lipid variation by WD in female mice, while there was little to no variation in the hippocampus, regardless of sex. On the other hand, lipid profiles were significantly affected by sex in all regions. Overall, 83 lipids were putatively identified in the mouse brain; among them, HexCer(40:1;O3) and PE(34:0) were found to have the largest statistical difference based on diet for female mice in the cortex and corpus callosum, respectively. Additional lipid changes are noted and can serve as a metric for understanding the brain's metabolomic response to changes in diet, particularly as it relates to disease.

Protein S -acylation is an important lipid modification and plays a series of biological functions. As a classic proteomic method for S -acylated proteome analysis, the acyl-biotin exchange and its derivative methods are known to be very labour-intensive and time-consuming all the time, and will result in significant sample loss. Multiple methanol-chloroform precipitations are involved in order to remove the substances that would interfere with enrichment and identification including detergents, the residual reduction and alkylation reagents. Here, we developed a rapid and convenient method for S -acylation proteomics by combining a dissolvable tube gel and the classic ABE method, a Dissolvable Gel based One-Tube sample Treatment method (DGOTT) method. The protein fixation rate, impact of the gel size on analysis performance and feasibility for analyzing complex samples were evaluated. This method enabled the alkylation and chemical substitution reactions to be conducted in a single EP tube, and convenient removal of interferents through gel washing, which could obviously simplify operations and shorten the sample treatment duration. Finally, we identified a total of 1625 potential S -acylated proteins from 800 μg of mouse brain cerebral cortex proteins. We believe that our method could offer potential for high-throughput analysis of protein S -acylation.

Data mining was performed at the databases of the Allen Institute for Brain Science (RRID:SCR_017001) searching for genes expressed selectively throughout the adult mouse mesocortex (transitional cortex ring predicted within the concentric ring theory of mammalian cortical structure, in contrast with central isocortex [ICx] and peripheral allocortex). We aimed to explore a shared molecular profile selective of all or most mesocortex areas. This approach checks and corroborates the precision of other previous definitory criteria, such as poor myelination and high kainate receptor level. Another aim was to examine which cortical areas properly belong to mesocortex. A total of 34 positive adult selective marker genes of mesocortex were identified, jointly with 12 negative selective markers, making a total of 46 markers. All of them identify the same set of cortical areas surrounding the molecularly different ICx as well as excluding adjacent allocortex. Four representative mesocortex markers-Crym, Lypd1, Cdh13, and Smoc2-are amply illustrated, jointly with complementary material including myelin basic protein, to check myelination, and Rorb, to check layer 4 presence. The retrosplenial (ReSp) area, long held to be mesocortical, does not share any of the 46 markers of mesocortex and instead expresses Nr4a2 and Tshz2, selective parahippocampal allocortex markers. Moreover, it is not hypomyelinic and lacks a Rorb-positive layer 4, aspects generally present in mesocortex. Exclusion of the ReSp area from the mesocortex ring reveals the latter to be closed at this locus instead by two adjacent areas previously thought to be associative visual ICx (reidentified here molecularly as postsplenial and parasplenial mesocortex areas). The concepts of ICx, mesocortex, and parahippocampal allocortex are thus subtly modified by substantial molecular evidence., (© 2024 The Author(s). The Journal of Comparative Neurology published by Wiley Periodicals LLC.)

Neuropeptides are key signaling molecules in the endocrine and nervous systems that regulate many critical physiological processes. Understanding the functions of neuropeptides in vivo requires the ability to monitor their dynamics with high specificity, sensitivity, and spatiotemporal resolution. However, this has been hindered by the lack of direct, sensitive, and noninvasive tools. We developed a series of GRAB (G protein-coupled receptor activation‒based) sensors for detecting somatostatin (SST), corticotropin-releasing factor (CRF), cholecystokinin (CCK), neuropeptide Y (NPY), neurotensin (NTS), and vasoactive intestinal peptide (VIP). These fluorescent sensors, which enable detection of specific neuropeptide binding at nanomolar concentrations, establish a robust tool kit for studying the release, function, and regulation of neuropeptides under both physiological and pathophysiological conditions.

Background: Important insights into the early pathogenesis of Alzheimer's disease can be provided by studies of autosomal dominant Alzheimer's disease and Down syndrome. However, it is unclear whether the timing and spatial distribution of amyloid accumulation differs between people with autosomal dominant Alzheimer's disease and those with Down syndrome. We aimed to directly compare amyloid changes between these two groups of people., Methods: In this cross-sectional study, we included participants (aged ≥25 years) with Down syndrome and sibling controls who had MRI and amyloid PET scans in the first data release (January, 2020) of the Alzheimer's Biomarker Consortium-Down Syndrome (ABC-DS) study. We also included carriers of autosomal dominant Alzheimer's disease genetic mutations and non-carrier familial controls who were within a similar age range to ABC-DS participants (25-73 years) and had MRI and amyloid PET scans at the time of a data freeze (December, 2020) of the Dominantly Inherited Alzheimer Network (DIAN) study. Controls from the two studies were combined into a single group. All DIAN study participants had genetic testing to determine PSEN1, PSEN2, or APP mutation status. APOE genotype was determined from blood samples. CSF samples were collected in a subset of ABC-DS and DIAN participants and the ratio of amyloid β42 (Aβ42) to Aβ40 (Aβ42/40) was measured to evaluate its Spearman's correlation with amyloid PET. Global PET amyloid burden was compared with regards to cognitive status, APOE ɛ4 status, sex, age, and estimated years to symptom onset. We further analysed amyloid PET deposition by autosomal dominant mutation type. We also assessed regional patterns of amyloid accumulation by estimated number of years to symptom onset. Within a subset of participants the relationship between amyloid PET and CSF Aβ42/40 was evaluated., Findings: 192 individuals with Down syndrome and 33 sibling controls from the ABC-DS study and 265 carriers of autosomal dominant Alzheimer's disease mutations and 169 non-carrier familial controls from the DIAN study were included in our analyses. PET amyloid centiloid and CSF Aβ42/40 were negatively correlated in carriers of autosomal dominant Alzheimer's disease mutations (n=216; r=-0·565; p<0·0001) and in people with Down syndrome (n=32; r=-0·801; p<0·0001). There was no difference in global PET amyloid burden between asymptomatic people with Down syndrome (mean 18·80 centiloids [SD 28·33]) versus asymptomatic mutation carriers (24·61 centiloids [30·27]; p=0·11) and between symptomatic people with Down syndrome (77·25 centiloids [41·76]) versus symptomatic mutation carriers (69·15 centiloids [51·10]; p=0·34). APOE ɛ4 status and sex had no effect on global amyloid PET deposition. Amyloid deposition was elevated significantly earlier in mutation carriers than in participants with Down syndrome (estimated years to symptom onset -23·0 vs -17·5; p=0·0002). PSEN1 mutations primarily drove this difference. Early amyloid accumulation occurred in striatal and cortical regions for both mutation carriers (n=265) and people with Down syndrome (n=128). Although mutation carriers had widespread amyloid accumulation in all cortical regions, the medial occipital regions were spared in people with Down syndrome., Interpretation: Despite minor differences, amyloid PET changes were similar between people with autosomal dominant Alzheimer's disease versus Down syndrome and strongly supported early amyloid dysregulation in individuals with Down syndrome. Individuals with Down syndrome aged at least 35 years might benefit from early intervention and warrant future inclusion in clinical trials, particularly given the relatively high incidence of Down syndrome., Funding: The National Institute on Aging, Riney and Brennan Funds, the Eunice Kennedy Shriver National Institute of Child Health and Human Development, the German Center for Neurodegenerative Diseases, and the Japan Agency for Medical Research and Development., Competing Interests: Declaration of interests TLSB has received funding from the National Institutes of Health and Siemens; has a licensing agreement from Sora Neuroscience but receives no financial compensation; has received honoraria for lectures, presentations, speakers bureaus, or educational events from Biogen and Eisai Genetech; has served on a scientific advisory board for Biogen; holds a leadership role in other board, society, committee, or advocacy groups for the American Society for Neuroradiology (unpaid) and Quantitative Imaging Biomarkers Alliance (unpaid); and has participated in radiopharmaceuticals and technology transfers with Avid Radiopharmaceuticals, Cerveau, and LMI. EMD received support from the National Institute on Aging, an anonymous organisation, the GHR Foundation, the DIAN-TU Pharma Consortium, Eli Lilly, and F Hoffmann La-Roche; has received speaking fees from Eisai and Eli Lilly; and is on the data safety and monitoring board and advisory boards of Eli Lilly, Alector, and Alzamend. WS has received research funding from the National Institute on Aging and the Eunice Kennedy Shriver National Institute of Child Health and Human Development. JPC serves as the chair of the American Neurological Association Dementia and Aging Special Interest Group and is on the medical advisory board of Humana Healthcare. CC has received consulting fees from GSK and Alector. AMF reports personal fees from Roche Diagnostics, Araclon/Grifols, and Diadem Research and grants from Biogen, outside the submitted work. BLH has received research funding from Roche and Autism Speaks; receives royalties from Oxford University Press for book publications; and is the chair of the data safety and monitoring board for the Department of Defense-funded study, “Comparative Effectiveness of EIBI and MABA”. BTC receives research funding from the National Institutes of Health. EH receives research funding from the National Institutes of Health and the BrightFocus Foundation. FL is supported by grants from the National Institute on Aging. HDR has received funding from the National Institutes of Health and is on the scientific advisory committee for the Hereditary Disease Foundation. JHL has received research funding from the National Institutes of Health and the National Institute on Aging. RJP receives research funding from the National Institutes of Health and the National Institute on Aging. RJB is Director of DIAN-TU and Principal Investigator of DIAN-TU001; receives research support from the National Institute on Aging of the National Institutes of Health, DIAN-TU trial pharmaceutical partners (Eli Lilly, F Hoffmann-La Roche, Janssen, Eisai, Biogen, and Avid Radiopharmaceuticals), the Alzheimer's Association, the GHR Foundation, an anonymous organisation, the DIAN-TU Pharma Consortium (active members Biogen, Eisai, Eli Lilly, Janssen, and F Hoffmann-La Roche/Genentech; previous members AbbVie, Amgen, AstraZeneca, Forum, Mithridion, Novartis, Pfizer, Sanofi, and United Neuroscience), the NfL Consortium (F Hoffmann-La Roche, Biogen, AbbVie, and Bristol Myers Squibb), and the Tau SILK Consortium (Eli Lilly, Biogen, and AbbVie); has been an invited speaker and consultant for AC Immune, F Hoffmann-La Roche, the Korean Dementia Association, the American Neurological Association, and Janssen; has been a consultant for Amgen, F Hoffmann-La Roche, and Eisai; and has submitted the US non-provisional patent application named “Methods for Measuring the Metabolism of CNS Derived Biomolecules In Vivo” and a provisional patent application named “Plasma Based Methods for Detecting CNS Amyloid Deposition”. BMA receives research funding from the National Institutes of Health and has a patent pending (“Markers of Neurotoxicity in CAR T patients”). MSR has received consulting fees from AC Immune, Embic, and Keystone Bio and has received research support from the National Institutes of Health, Avid, Baxter, Eisai, Elan, Genentech, Janssen, Lilly, Merck, and Roche. All other authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: The brain endocannabinoid system is believed to play significant roles in anti-nociception, fear response, anxiety, and stress. This study investigated the effects of rat inguinal surgery on the levels of endocannabinoids in the cerebral cortex., Aim: The aim of this study was to investigate the effects of acute post-surgical pain on the levels of endocannabinoids in the cerebral cortex., Methods: Quantitation of endocannabinoids in the rat cerebral cortex was performed by liquid chromatography-tandem mass spectrometry., Results: There was no significant difference in the cerebral cortical levels of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) between the sham and surgery experimental groups. However, there were lateralized differences in the levels of these endocannabinoids between the right and left cerebral cortices irrespective of the two groups. The concentrations of AEA and 2-AG were significantly higher in the right cerebral cortex compared to the contralateral cerebral cortex., Conclusion: Acute post-surgical pain did not induce significant alterations in the cerebral cortical levels of endocannabinoids in this study, but the phenomenon of lateralization of the cerebral cortical AEA and 2-AG levels was observed; this latter finding may be related to the role played by endocannabinoids in fear conditioning.

Neuronal remodeling after brain injury is essential for functional recovery. After unilateral cortical lesion, axons from the intact cortex ectopically project to the denervated midbrain, but the molecular mechanisms remain largely unknown. To address this issue, we examined gene expression profiles in denervated and intact mouse midbrains after hemispherectomy at early developmental stages using mice of either sex, when ectopic contralateral projection occurs robustly. The analysis showed that various axon growth-related genes were upregulated in the denervated midbrain, and most of these genes are reportedly expressed by glial cells. To identify the underlying molecules, the receptors for candidate upregulated molecules were knocked out in layer 5 projection neurons in the intact cortex, using the CRISPR/Cas9-mediated method, and axonal projection from the knocked-out cortical neurons was examined after hemispherectomy. We found that the ectopic projection was significantly reduced when integrin subunit β three or neurotrophic receptor tyrosine kinase 2 (also known as TrkB) was knocked out. Overall, the present study suggests that denervated midbrain-derived glial factors contribute to lesion-induced remodeling of the cortico-mesencephalic projection via these receptors. SIGNIFICANCE STATEMENT After brain injury, compensatory neural circuits are established that contribute to functional recovery. However, little is known about the intrinsic mechanism that underlies the injury-induced remodeling. We found that after unilateral cortical ablation expression of axon-growth promoting factors is elevated in the denervated midbrain and is involved in the formation of ectopic axonal projection from the intact cortex. Evidence further demonstrated that these factors are expressed by astrocytes and microglia, which are activated in the denervated midbrain. Thus, our present study provides a new insight into the mechanism of lesion-induced axonal remodeling and further therapeutic strategies after brain injury., (Copyright © 2022 the authors.)

Network dynamics have been proposed as a mechanistic substrate for the information transfer across cortical and hippocampal circuits. However, little is known about the mechanisms that synchronize and coordinate these processes across widespread brain regions during offline states. Here we address the hypothesis that breathing acts as an oscillatory pacemaker, persistently coupling distributed brain circuit dynamics. Using large-scale recordings from a number of cortical and subcortical brain regions in behaving mice, we uncover the presence of an intracerebral respiratory corollary discharge, that modulates neural activity across these circuits. During offline states, the respiratory modulation underlies the coupling of hippocampal sharp-wave ripples and cortical DOWN/UP state transitions, which mediates systems memory consolidation. These results highlight breathing, a perennial brain rhythm, as an oscillatory scaffold for the functional coordination of the limbic circuit that supports the segregation and integration of information flow across neuronal networks during offline states., (© 2022. The Author(s).)

The aim of our investigation was to make a comparative assessment of the biological effects of silver nanoparticles encapsulated in a natural and synthetic polymer matrix. We carried out a comparative assessment of the biological effect of silver nanocomposites on natural (arabinogalactan) and synthetic (poly-1-vinyl-1,2,4-triazole) matrices. We used 144 three-month-old white outbred male rats, which were divided into six groups. Substances were administered orally for 9 days at a dose 500 μg/kg. Twelve rats from each group were withdrawn from the experiment immediately after nine days of exposure (early period), and the remaining 12 rats were withdrawn from the experiment 6 months after the end of the nine-day exposure (long-term period). We investigated the parietal-temporal area of the cerebral cortex using histological (morphological assessments of nervous tissue), electron microscopic (calculation of mitochondrial areas and assessment of the quality of the cell nucleus), and immunohistochemical methods (study of the expression of proteins regulating apoptosis bcl-2 and caspase 3). We found that the effect of the nanocomposite on the arabinogalactan matrix causes a disturbance in the nervous tissue structure, an increase in the area of mitochondria, a disturbance of the structure of nerve cells, and activation of the process of apoptosis.

Characterizing cortex-wide neural activity is essential for understanding large-scale interactions among distributed cortical regions. Here, we describe a protocol using wide-field calcium imaging to monitor the cortex-wide activity in awake, head-fixed mice. This approach provides sufficient signal-to-noise ratio and spatiotemporal resolution to capture large-scale neural activity in behaving mice on a moment-by-moment basis. The use of genetically encoded calcium indicators allows longitudinal imaging over months and can achieve cell-type specificity. We also describe a pipeline to process the imaging data. For complete details on the use and execution of this protocol, please refer to Makino et al. (2017) and Liu et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

We examined the number, distribution, and immunoreactivity of the infracortical white matter neuronal population, also termed white matter interstitial cells (WMICs), throughout the telencephalic white matter of an adult female chimpanzee. Staining for neuronal nuclear marker (NeuN) revealed WMICs throughout the infracortical white matter, these cells being most numerous and dense close to the inner border of cortical layer VI, decreasing significantly in density with depth in the white matter. Stereological analysis of NeuN-immunopositive cells revealed an estimate of approximately 137.2 million WMICs within the infracortical white matter of the chimpanzee brain studied. Immunostaining revealed subpopulations of WMICs containing neuronal nitric oxide synthase (nNOS, approximately 14.4 million in number), calretinin (CR, approximately 16.7 million), very few WMICs containing parvalbumin (PV), and no calbindin-immunopositive neurons. The nNOS, CR, and PV immunopositive WMICs, possibly all inhibitory neurons, represent approximately 22.6% of the total WMIC population. As the white matter is affected in many cognitive conditions, such as schizophrenia, autism, epilepsy, and also in neurodegenerative diseases, understanding these neurons across species is important for the translation of findings of neural dysfunction in animal models to humans. Furthermore, studies of WMICs in species such as apes provide a crucial phylogenetic context for understanding the evolution of these cell types in the human brain., (© 2021 Wiley Periodicals LLC.)

Protein glycosylation is crucial for the central nervous system and brain functions, including processes that are defective in Alzheimer disease (AD) such as neurogenesis, synaptic function, and memory formation. Still, the roles of glycans in the development of AD are relatively unexplored. Glycomics studies of cerebrospinal fluid (CSF) have previously shown altered glycosylation pattern in patients with different stages of cognitive impairment, including AD, compared to healthy controls. As a consequence, we hypothesized that the glycan profile is altered in the brain of patients with AD and analyzed the asparagine-linked (N-linked) glycan profile in hippocampus and cortex in AD and control brain. Glycans were enzymatically liberated from brain glycoproteins and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Eleven glycans showed significantly different levels in hippocampus compared to cortex in both control and AD brain. Two glycans in cortex and four in hippocampus showed different levels in AD compared to control brain. All glycans that differed between controls and AD brain had similar structures with one sialic acid, at least one fucose and a confirmed or potential bisecting N-acetylglucosamine (GlcNAc). The glycans that were altered in AD brain differed from those that were altered in AD CSF. One glycan found to be present in significantly lower levels in both hippocampus and cortex in AD compared to control contained a structurally and functionally interesting epitope that we assign as a terminal galactose decorated with fucose and sialic acid. Altogether, these studies suggest that protein glycosylation is an important component in the development of AD and warrants further studies., (© 2020 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

One of the urgent tasks of neuroscience is to understand how neuronal circuits operate, what makes them fail, and how to repair them when needed. Achieving this goal requires identifying the principal circuitry elements and their interactions with one another. However, what constitutes 'an atom' of a neuronal circuit, a neuronal type, is a complex question. In this review we focus on a class of cortical neurons that are exclusively identified by the expression of vasoactive intestinal polypeptide (VIP) and choline acetyltransferase (ChAT). The genetic profile of these VIP + /ChAT + interneurons suggests that they can release both γ-aminobutyric acid (GABA) and acetylcholine (ACh). This hints to a specific potential role in the cortical circuitry. Yet the VIP + /ChAT + interneurons are sparse (a mere 0.5% of the cortical neurons), which raises questions about their potential to significantly affect the circuit function. In view of recent developments in genetic techniques that allow for direct manipulation of these neurons, we provide a thorough and updated picture of the properties of the VIP + /ChAT + interneurons. We discuss their genetic profile, their physiological and structural properties, and their input-output mapping in sensory cortices and the medial prefrontal cortex (mPFC). Then, we examine possible amplification mechanisms for mediating their function in the cortical microcircuit. Finally, we discuss directions for further exploration of the VIP + /ChAT + population, focusing on its function during behavioral tasks as compared to the VIP + /ChAT - population., (© 2020 International Society for Neurochemistry.)

We studied the effect of various detergents (Tween-20, Triton X-100, and sodium deoxycholate) on activity and magnesium-dependent properties of Na + ,K + -ATPase of the crude membrane fraction of rat cerebral cortex. All studied detergents significantly increased activity of the studied enzyme in a concentration-dependent manner. Sodium deoxycholate provided significantly higher values Na + ,K + -ATPase activity (by ≈50%) than Triton X-100 and Tween-20. In the presence of Triton X-100, a changed pattern of the dependence of enzyme activity on the concentration of magnesium ions in the incubation solution was noted. Separate measurement of activities of Na + ,K + -ATPase isoforms made it possible to assume that changes in magnesium-dependent properties are due to the predominant effect of Triton X-100 on ouabain-sensitive α2- and α3-isoforms., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Intrinsic optical signal imaging (ISI) is a hemodynamic response-based technique to map the functional architecture of the cortex. ISI is often used as an auxiliary method to localize cortical areas for targeted electrophysiology, pharmacology, or imaging experiments. Here, we provide a protocol for ISI through a cranial window with an access port to identify the area of the primary visual cortex (V1) in a head-fixed mouse, followed by targeted viral vector injection, which enables subsequent two-photon imaging of V1 layer 6 corticothalamic neurons. For complete details on the use and execution of this protocol, please refer to our paper Augustinaite and Kuhn (2020b)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

This study was the first comprehensive investigation of the dependence of mitochondrial enzyme response (catalytic subunits of mitochondrial complexes (MC) I-V, including NDUFV2, SDHA, Cyt b, COX1 and ATP5A) and mitochondrial ultrastructure in the rat cerebral cortex (CC) on the severity and duration of in vivo hypoxic exposures. The role of individual animal's resistance to hypoxia was also studied. The respiratory chain (RC) was shown to respond to changes in environmental [O 2 ] as follows: (a) differential reaction of mitochondrial enzymes, which depends on the severity of the hypoxic exposure and which indicates changes in the content and catalytic properties of mitochondrial enzymes, both during acute and multiple exposures; and (b) ultrastructural changes in mitochondria, which reflect various degrees of mitochondrial energization. Within a specific range of reduced O 2 concentrations, activation of the MC II is a compensatory response supporting the RC electron transport function. In this process, MC I develops new kinetic properties, and its function recovers in hypoxia by reprograming the RC substrate site. Therefore, the mitochondrial RC performs as an in vivo molecular oxygen sensor. Substantial differences between responses of rats with high and low resistance to hypoxia were determined.

The developing human brain is uniquely vulnerable to methylmercury (MeHg) resulting in lasting effects especially in developing cortical structures. Here we assess by single-cell RNA sequencing (scRNAseq) persistent effects of developmental MeHg exposure in a differentiating cortical human-induced pluripotent stem cell (hiPSC) model which we exposed to in vivo relevant and non-cytotoxic MeHg (0.1 and 1.0 μM) concentrations. The cultures were exposed continuously for 6 days either once only during days 4-10, a stage representative of neural epithelial- and radial glia cells, or twice on days 4-10 and days 14-20, a somewhat later stage which includes intermediate precursors and early postmitotic neurons. After the completion of MeHg exposure the cultures were differentiated further until day 38 and then assessed for persistent MeHg-induced effects by scRNAseq. We report subtle, but significant changes in the population size of different cortical cell types/stages and cell cycle. We also observe MeHg-dependent differential gene expression and altered biological processes as determined by Gene Ontology analysis. Our data demonstrate that MeHg results in changes in gene expression in human developing cortical neurons that manifest well after cessation of exposure and that these changes are cell type-, developmental stage-, and exposure paradigm-specific., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Amyloid plaques are a hallmark of Alzheimer's disease (AD) that develop in its earliest stages. Thus, non-invasive detection of these plaques would be invaluable for diagnosis and the development and monitoring of treatments, but this remains a challenge due to their small size. Here, we investigated the utility of manganese-enhanced MRI (MEMRI) for visualizing plaques in transgenic rodent models of AD across two species: 5xFAD mice and TgF344-AD rats. Animals were given subcutaneous injections of MnCl 2 and imaged in vivo using a 9.4 T Bruker scanner. MnCl 2 improved signal-to-noise ratio but was not necessary to detect plaques in high-resolution images. Plaques were visible in all transgenic animals and no wild-types, and quantitative susceptibility mapping showed that they were more paramagnetic than the surrounding tissue. This, combined with beta-amyloid and iron staining, indicate that plaque MR visibility in both animal models was driven by plaque size and iron load. Longitudinal relaxation rate mapping revealed increased manganese uptake in brain regions of high plaque burden in transgenic animals compared to their wild-type littermates. This was limited to the rhinencephalon in the TgF344-AD rats, while it was most significantly increased in the cortex of the 5xFAD mice. Alizarin Red staining suggests that manganese bound to plaques in 5xFAD mice but not in TgF344-AD rats. Multi-parametric MEMRI is a simple, viable method for detecting amyloid plaques in rodent models of AD. Manganese-induced signal enhancement can enable higher-resolution imaging, which is key to visualizing these small amyloid deposits. We also present the first in vivo evidence of manganese as a potential targeted contrast agent for imaging plaques in the 5xFAD model of AD.

Wernicke-Korsakoff Syndrome (WKS) is a neuropsychiatric disorder whose etiology is a thiamine deficiency (TD), with alcoholism being the main underlying cause. Previous evidence suggests the presence of initial neuroinflammation and oxidative/nitrosative stress in the physiopathology, although the specific molecular mechanisms underlying TD-induced brain damage and behavioral disabilities are unknown. We explored the specific role of the innate immune receptor TLR4 in three murine models of WKS, based on the combination of a thiamine-deficient diet and pyrithiamine injections (0.25 mg/kg, i.p.) over time. The Symptomatic Model (SM) allowed us to describe the complete neurological/neurobehavioral symptomatology over 16 days of TD. Animals showed an upregulation of the TLR4 signaling pathway both in the frontal cortex (FC) and cerebellum and clear motor impairments related with cerebellar dysfunction. However, in the Pre-Symptomatic Model (PSM), 12 days of TD induced the TLR4 pathway upregulation in the FC, which correlated with disinhibited-like behavior, but not in the cerebellum, and no motor impairments. In addition, we tested the effects of the biolipid oleoylethanolamide (OEA, 10 mg/kg, i.p., once daily, starting before any symptom of the pathology is manifested) through the Glucose-Precipitated Model (GPM), which was generated by glucose loading (5 g/kg, i.v., last day) in thiamine-deficient animals to accelerate damage. Pretreatment with OEA prevented the TLR4-induced signature in the FC, as well as an underlying incipient memory disability and disinhibited-like behavior. This study suggests a key role for TLR4 in TD-induced neuroinflammation in the FC and cerebellum, and it reveals different vulnerability of these brain regions in WKS over time. Pre-treatment with OEA counteracts TD-induced TLR4-associated neuroinflammation and may serve as co-adjuvant therapy to prevent WKS-induced neurobehavioral alterations., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Emerging evidence suggests that amino acid (AA) neurotransmitters play important roles in the pathophysiological processes of cerebral ischemia. In this work, an HPLC with fluorescence detection (HPLC-FLR) method was developed for the simultaneous determination of 18 AAs in the cortex and plasma after cerebral ischemia in mice. The ischemia model was prepared by bilateral common carotid artery occlusion, and then the cortex and plasma of the sham, ischemia, and naringenin groups were collected. Based on the protein precipitation method, a simple and effective sample preparation method was developed. The treated sample contained minimal proteins and lipids. The analysis of the sample was performed by the proposed HPLC-FLR method in combination with o-phthalaldehyde. The results showed a statistically significant increase in excitatory AAs (aspartic acid and glutamic acid), inhibitory AAs (glycine and 4-aminobutyric acid), phenylalanine, citrulline, isoleucine, and leucine levels, and a decrease of glutathione and phenylalanine levels when compared with the sham group in the cortex. Besides, the administration of naringenin had significant effects on excitatory AAs, inhibitory AA (glycine), glutamine, tyrosine, phenylalanine, and leucine levels when compared with the sham group in the cortex. These findings could be utilized in studying and clarifying the mechanisms of ischemia., (© 2021 John Wiley & Sons, Ltd.)

Many developmental syndromes have been linked to genetic mutations that cause abnormal ERK/MAPK activity; however, the neuropathological effects of hyperactive signaling are not fully understood. Here, we examined whether hyperactivation of MEK1 modifies the development of GABAergic cortical interneurons (CINs), a heterogeneous population of inhibitory neurons necessary for cortical function. We show that GABAergic-neuron specific MEK1 hyperactivation in vivo leads to increased cleaved caspase-3 labeling in a subpopulation of immature neurons in the embryonic subpallial mantle zone. Adult mutants displayed a significant loss of parvalbumin (PV), but not somatostatin, expressing CINs and a reduction in perisomatic inhibitory synapses on excitatory neurons. Surviving mutant PV-CINs maintained a typical fast-spiking phenotype but showed signs of decreased intrinsic excitability that coincided with an increased risk of seizure-like phenotypes. In contrast to other mouse models of PV-CIN loss, we discovered a robust increase in the accumulation of perineuronal nets, an extracellular structure thought to restrict plasticity. Indeed, we found that mutants exhibited a significant impairment in the acquisition of behavioral response inhibition capacity. Overall, our data suggest PV-CIN development is particularly sensitive to hyperactive MEK1 signaling, which may underlie certain neurological deficits frequently observed in ERK/MAPK-linked syndromes., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

The high-mobility group box-1 (HMGB1) protein is a transcription-regulating protein located in the nucleus. However, it serves as a damage-associated molecular pattern protein that activates immune cells and stimulates inflammatory cytokines to accentuate neuroinflammation after release from damaged cells. In contrast, Inter-alpha Inhibitor Proteins (IAIPs) are proteins with immunomodulatory effects including inhibition of pro-inflammatory cytokines. We have demonstrated that IAIPs exhibit neuroprotective properties in neonatal rats exposed to hypoxic-ischemic (HI) brain injury. In addition, previous studies have suggested that the light chain of IAIPs, bikunin, may exert its anti-inflammatory effects by inhibiting HMGB1 in a variety of different injury models in adult subjects. The objectives of the current study were to confirm whether HMGB1 is a target of IAIPs by investigating the potential binding characteristics of HMGB1 and IAIPs in vitro, and co-localization in vivo in cerebral cortices after exposure to HI injury. Solid-phase binding assays and surface plasmon resonance (SPR) were used to determine the physical binding characteristics between IAIPs and HMGB1. Cellular localizations of IAIPs-HMGB1 in neonatal rat cortex were visualized by double labeling with anti-IAIPs and anti-HMGB1 antibodies. Solid-phase binding and SPR demonstrated specific binding between IAIPs and HMGB1 in vitro. Cortical cytoplasmic and nuclear co-localization of IAIPs and HMGB1 were detected by immunofluorescent staining in control and rats immediately and 3 hours after HI. In conclusion, HMGB1 and IAIPs exhibit direct binding in vitro and co-localization in vivo in neonatal rats exposed to HI brain injury suggesting HMGB1 could be a target of IAIPs., (© 2021 Federation of American Societies for Experimental Biology.)

Astrocytes are major producers of the extracellular matrix (ECM), which is involved in the plasticity of the developing brain. In utero alcohol exposure alters neuronal plasticity. Glycosaminoglycans (GAGs) are a family of polysaccharides present in the extracellular space; chondroitin sulfate (CS)- and heparan sulfate (HS)-GAGs are covalently bound to core proteins to form proteoglycans (PGs). Hyaluronic acid (HA)-GAGs are not bound to core proteins. In this study we investigated the contribution of astrocytes to CS-, HS-, and HA-GAG production by comparing the makeup of these GAGs in cortical astrocyte cultures and the neonatal rat cortex. We also explored alterations induced by ethanol in GAG and core protein levels in astrocytes. Finally, we investigated the relative expression in astrocytes of CS-PGs of the lectican family of proteins, major components of the brain ECM, in vivo using translating ribosome affinity purification (TRAP) (in Aldh1l1-EGFP-Rpl10a mice. Cortical astrocytes produce low levels of HA and show low expression of genes involved in HA biosynthesis compared to the whole developing cortex. Astrocytes have high levels of chondroitin-0-sulfate (C0S)-GAGs (possibly because of a higher sulfatase enzyme expression) and HS-GAGs. Ethanol upregulates C4S-GAGs as well as brain-specific lecticans neurocan and brevican, which are highly enriched in astrocytes of the developing cortex in vivo. These results begin to elucidate the role of astrocytes in the biosynthesis of CS- HS- and HA-GAGs, and suggest that ethanol-induced alterations of neuronal development may be in part mediated by increased astrocyte GAG levels and neurocan and brevican expression.

The parabrachial nucleus (PB) is a complex structure located at the junction of the midbrain and hindbrain. Its neurons have diverse genetic profiles and influence a variety of homeostatic functions. While its cytoarchitecture and overall efferent projections are known, we lack comprehensive information on the projection patterns of specific neuronal subtypes in the PB. In this study, we compared the projection patterns of glutamatergic neurons here with a subpopulation expressing the transcription factor Foxp2 and a further subpopulation expressing the neuropeptide Pdyn. To do this, we injected an AAV into the PB region to deliver a Cre-dependent anterograde tracer (synaptophysin-mCherry) in three different strains of Cre-driver mice. We then analyzed 147 neuroanatomical regions for labeled boutons in every brain (n = 11). Overall, glutamatergic neurons in the PB region project to a wide variety of sites in the cerebral cortex, basal forebrain, bed nucleus of the stria terminalis, amygdala, diencephalon, and brainstem. Foxp2 and Pdyn subpopulations project heavily to the hypothalamus, but not to the cortex, basal forebrain, or amygdala. Among the few differences between Foxp2 and Pdyn cases was a notable lack of Pdyn projections to the ventromedial hypothalamic nucleus. Our results indicate that genetic identity determines connectivity (and therefore, function), providing a framework for mapping all PB output projections based on the genetic identity of its neurons. Using genetic markers to systematically classify PB neurons and their efferent projections will enhance the translation of research findings from experimental animals to humans., (© 2020 Wiley Periodicals LLC.)

Astrocytes, a highly heterogeneous population of glial cells, serve as essential regulators of brain development and homeostasis. The heterogeneity of astrocyte populations underlies the diversity in their functions. In addition to the typical mammalian astrocyte architecture, the cerebral cortex of humans exhibits a radial distribution of interlaminar astrocytes in the supragranular layers. These primate-specific interlaminar astrocytes are located in the superficial layer and project long processes traversing multiple layers of the cerebral cortex. However, due to the lack of accessible experimental models, their functional properties and their role in regulating neuronal circuits remain unclear. Here we modeled human interlaminar astrocytes in humanized glial chimeric mice by engrafting astrocytes differentiated from human-induced pluripotent stem cells into the mouse cortex. This model provides a novel platform for understanding neuron-glial interaction and its alterations in neurological diseases., (© 2020 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals LLC.)

Combination of ischemia and β-amyloid (Aβ) toxicity has been shown to simultaneously increase neuro-inflammation, endogenous Aβ deposition, and neurodegeneration. However, studies on the evolution of infarct and panorama of cellular degeneration as a synergistic or overlapping mechanism between ischemia and Aβ toxicity are lacking. Here, we compared fluorojade B (FJB) and hematoxylin and eosin (H&E) stains primarily to examine the chronology of infarct, and the viability and morphological changes in neuroglia and neurons located in different brain regions on d1, d7, and d28 post Aβ toxicity and endothelin-1 induced ischemia (ET1) in rats. We demonstrated a regional difference in cellular degeneration between cortex, corpus callosum, striatum, globus pallidus, and thalamus after cerebral injury. Glial cells in the cortex and corpus callosum underwent delayed FJB staining from d7 to d28, but neurons in cortex disappeared within the first week of cerebral injury. Striatal lesion core and globus pallidus of Aβ + ET1 rats showed extensive degeneration of neuronal cells compared with ET1 rats alone starting from d1. Differential and exacerbated expressions of cyclooxygenase-2 might be the cause of excessive neuronal demise in the striatum of Aβ + ET1 rats. Such an investigation may improve our understanding to identify and manipulate a critical therapeutic window post comorbid injury., (© 2020 Wiley Periodicals LLC.)

Amyloid-β (Aβ) fibrils exhibit self-propagating, molecular-level polymorphisms that may contribute to variations in clinical and pathological characteristics of Alzheimer's disease (AD). We report the molecular structure of a specific fibril polymorph, formed by 40-residue Aβ peptides (Aβ40), that is derived from cortical tissue of an AD patient by seeded fibril growth. The structure is determined from cryogenic electron microscopy (cryoEM) images, supplemented by mass-per-length (MPL) measurements and solid-state NMR (ssNMR) data. Previous ssNMR studies with multiple AD patients had identified this polymorph as the most prevalent brain-derived Aβ40 fibril polymorph from typical AD patients. The structure, which has 2.8-Å resolution according to standard criteria, differs qualitatively from all previously described Aβ fibril structures, both in its molecular conformations and its organization of cross-β subunits. Unique features include twofold screw symmetry about the fibril growth axis, despite an MPL value that indicates three Aβ40 molecules per 4.8-Å β-sheet spacing, a four-layered architecture, and fully extended conformations for molecules in the central two cross-β layers. The cryoEM density, ssNMR data, and MPL data are consistent with β-hairpin conformations for molecules in the outer cross-β layers. Knowledge of this brain-derived fibril structure may contribute to the development of structure-specific amyloid imaging agents and aggregation inhibitors with greater diagnostic and therapeutic utility., Competing Interests: The authors declare no competing interest.

Excessive brain iron negatively affects working memory and related processes but the impact of cortical iron on task-relevant, cortical brain networks is unknown. We hypothesized that high cortical iron concentration may disrupt functional circuitry within cortical networks supporting working memory performance. Fifty-five healthy older adults completed an N-Back working memory paradigm while functional magnetic resonance imaging (fMRI) was performed. Participants also underwent quantitative susceptibility mapping (QSM) imaging for assessment of non-heme brain iron concentration. Additionally, pseudo continuous arterial spin labeling scans were obtained to control for potential contributions of cerebral blood volume and structural brain images were used to control for contributions of brain volume. Task performance was positively correlated with strength of task-based functional connectivity (tFC) between brain regions of the frontoparietal working memory network. However, higher cortical iron concentration was associated with lower tFC within this frontoparietal network and with poorer working memory performance after controlling for both cerebral blood flow and brain volume. Our results suggest that high cortical iron concentration disrupts communication within frontoparietal networks supporting working memory and is associated with reduced working memory performance in older adults., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2020. Published by Elsevier Inc.)

Astrocytes are actively involved in a neuroprotective role in the brain, which includes scavenging reactive oxygen species to minimize tissue damage. They also modulate neuroinflammation and reactive gliosis prevalent in several brain disorders like epilepsy, Alzheimer's, and Parkinson's disease. In animal models, targeted manipulation of astrocytic function via modulation of their calcium (Ca 2+ ) oscillations by incorporating light-sensitive cation channels like Channelrhodopsin-2 (ChR2) offers a promising avenue in influencing the long-term progression of these disorders. However, using adult animals for Ca 2+ imaging poses major challenges, including accelerated deterioration of in situ slice health and age- related changes. Additionally, optogenetic preparations necessitate usage of a red-shifted Ca 2+ indicator like Rhod-2 AM to avoid overlapping light issues between ChR2 and the Ca 2+ indicator during simultaneous optogenetic stimulation and imaging. In this article, we provide an experimental setting that uses live adult murine brain slices (2-5 months) from a knock-in model expressing Channelrhodopsin-2 (ChR2(C128S)) in cortical astrocytes, loaded with Rhod-2 AM to elicit robust Ca 2+ response to light stimulation. We have developed and standardized a protocol for brain extraction, sectioning, Rhod-2 AM loading, maintenance of slice health, and Ca 2+ imaging during light stimulation. This has been successfully applied to optogenetically control adult cortical astrocytes, which exhibit synchronous patterns of Ca 2+ activity upon light stimulation, drastically different from resting spontaneous activity. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Experimental preparation, setup, slice preparation and Rhod-2 AM staining Basic Protocol 2: Image acquisition and analysis., (© 2020 Wiley Periodicals LLC.)

JLX001, a new dihydrochloride of Cyclovirobuxine D (CVB-D), has bioactivities against ischemia injury. The blood-brain barrier (BBB) disruption is involved in the pathogeneses of ischemic stroke. This study was designed to explore the effect and potential mechanism of JLX001 on the BBB after ischemic stroke. Rats were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) to mimic cerebral ischemia in vivo. In vitro, rat primary brain microvascular endothelial cells (PBMECs) were cultured and exposed to oxygen-glucose deprivation/reoxygenation (OGD/R). Posttreatment of JLX001 for 15 days after MCAO/R improved the behavior, learning and memory ability. Pretreatment of JLX001 for 3 days significantly attenuated infarct volume, lessened brain edema, mitigated BBB disruption and decreased the neurological deficit score in MCAO/R rats. Moreover, JLX001 increased cell viability and reduced sodium fluorescein leakage after OGD/R injury. In addition, JLX001 increased the expressions of Claudin-5 and Occludin, decreased the expression of MMP-9 both in vivo and in vitro. Moreover, immunofluorescence staining and western immunoblotting results showed that JLX001 increased the expressions of tight junction proteins via activating Wnt/β-catenin signal pathway in vivo and in vitro, which may be associated with the activation of PI3K/Akt signaling. Besides, XAV939 (an inhibitor of the Wnt/β-catenin pathway) proved the connection of JLX001 and Wnt/β-catenin pathway. These results suggest that JLX001 alleviates BBB disruption after MCAO/R and OGD/R possibly by alleviating MMP-9 and activating the Wnt/β-catenin signaling pathway., (Copyright © 2020. Published by Elsevier Inc.)

Electrical stimulation has been critical in the development of an understanding of brain function and disease. Despite its widespread use and obvious clinical potential, the mechanisms governing stimulation in the cortex remain largely unexplored in the context of pulse parameters. Modeling studies have suggested that modulation of stimulation pulse waveform may be able to control the probability of neuronal activation to selectively stimulate either cell bodies or passing fibers depending on the leading polarity. Thus, asymmetric waveforms with equal charge per phase (i.e., increasing the leading phase duration and proportionately decreasing the amplitude) may be able to activate a more spatially localized or distributed population of neurons if the leading phase is cathodic or anodic, respectively. Here, we use two-photon and mesoscale calcium imaging of GCaMP6s expressed in excitatory pyramidal neurons of male mice to investigate the role of pulse polarity and waveform asymmetry on the spatiotemporal properties of direct neuronal activation with 10-Hz electrical stimulation. We demonstrate that increasing cathodic asymmetry effectively reduces neuronal activation and results in a more spatially localized subpopulation of activated neurons without sacrificing the density of activated neurons around the electrode. Conversely, increasing anodic asymmetry increases the spatial spread of activation and highly resembles spatiotemporal calcium activity induced by conventional symmetric cathodic stimulation. These results suggest that stimulation polarity and asymmetry can be used to modulate the spatiotemporal dynamics of neuronal activity thus increasing the effective parameter space of electrical stimulation to restore sensation and study circuit dynamics., (© 2020 Wiley Periodicals, Inc.)

The EAG ( ether-à-go-go ) family of voltage-gated K + channels are important regulators of neuronal and cardiac action potential firing (excitability) and have major roles in human diseases such as epilepsy, schizophrenia, cancer, and sudden cardiac death. A defining feature of EAG (Kv10-12) channels is a highly conserved domain on the N terminus, known as the eag domain, consisting of a Per-ARNT-Sim (PAS) domain capped by a short sequence containing an amphipathic helix (Cap domain). The PAS and Cap domains are both vital for the normal function of EAG channels. Using heme-affinity pulldown assays and proteomics of lysates from primary cortical neurons, we identified that an EAG channel, hERG3 (Kv11.3), binds to heme. In whole-cell electrophysiology experiments, we identified that heme inhibits hERG3 channel activity. In addition, we expressed the Cap and PAS domain of hERG3 in Escherichia coli and, using spectroscopy and kinetics, identified the PAS domain as the location for heme binding. The results identify heme as a regulator of hERG3 channel activity. These observations are discussed in the context of the emerging role for heme as a regulator of ion channel activity in cells., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Burton et al.)

Alzheimer's disease (AD) is a common neurodegenerative disease that is always accompanied by synaptic loss in the brain. Safflower yellow (SY) is the extract of safflower, a traditional Chinese medicine, which has shown neuroprotective effects in recent studies. However, the mechanism of SY in protecting synapses remains unclear. In this study, we are going to study the mechanism of how SY treats AD in terms of synaptic plasticity. We found, via behavioral experiments, that SY treatment could improve the abilities of learning and memory in APP/PS1 mice. In addition, using Golgi staining and HE staining, we found that SY treatment could reduce the loss of dendritic spines in the pathological condition and could maintain the normal physiological state of the cells in cortex and in hippocampus. In addition, the results of immunofluorescence staining and western blotting showed that SY treatment could significantly increase the expression of synapse-related proteins. Moreover, after being treated with SY, the expression of iNOS (marker of M1 microglia) declined remarkably, and the level of Arginase-1 (marker of M2 microglia) increased significantly. Finally, we found BDNF/TrkB/ERK signaling cascade was activated. These results indicate that SY enhances synaptic plasticity in APP/PS1 mice by regulating microglia activation phenotypes and BDNF/TrkB/ERK signaling pathway.

Fourier Transform Infrared microspectroscopy (μFTIR) is a very useful method to analyze the biochemical properties of biological samples in situ. Many diseases affecting the central nervous system (CNS) have been studied using this method, to elucidate alterations in lipid oxidation or protein aggregation, among others. In this work, we describe in detail the characteristics between grey matter (GM) and white matter (WM) areas of the human brain by μFTIR, and we compare them with the mouse brain (strain C57BL/6), the most used animal model in neurological disorders. Our results show a clear different infrared profile between brain areas in the lipid region of both species. After applying a second derivative in the data, we established a 1.5 threshold value for the lipid/protein ratio to discriminate between GM and WM areas in non-pathological conditions. Furthermore, we demonstrated intrinsic differences of lipids and proteins by cerebral area. Lipids from GM present higher C=CH, C=O and CH 3 functional groups compared to WM in humans and mice. Regarding proteins, GM present lower Amide II amounts and higher intramolecular β-sheet structure amounts with respect to WM in both species. However, the presence of intermolecular β-sheet structures, which is related to β-aggregation, was only observed in the GM of some human individuals. The present study defines the relevant biochemical properties of non-pathological human and mouse brains by μFTIR as a benchmark for future studies involving CNS pathological samples.

The cerebral cortex, with all its computational power, can only influence behavior via corticofugal connections originating from layer 5 (L5) cells (Sherman and Guillery, 2013). To begin to establish the global pattern of these outputs, we examined L5 efferents originating from four cortical areas: somatosensory, visual, motor, and prefrontal (i.e., ventromedial orbitofrontal) cortex. We injected Cre-dependent adeno-associated virus in an Rbp4 -Cre transgenic mouse line (both sexes) to label these L5 efferents selectively. Our study reveals that, across this diverse series of cortical regions, L5 commonly projects to multiple thalamic and extrathalamic sites. We also identified several novel corticofugal targets (i.e., the lateral dorsal nucleus, submedial nucleus) previously unidentified as L5 targets. We identified common patterns for these projections: all areas innervated both thalamus and the midbrain, and all areas innervated multiple thalamic targets, including those with core and matrix cell types (Jones, 1998). An examination of the terminal size within each of these targets suggests that terminal populations of L5 efferents are not consistently large but vary with cortical area and target; and in some cases, these include small terminals only. Overall, our data reveal more widespread and diverse L5 efferents than previously appreciated, suggesting a generalizable role for this cortical layer in influencing motor commands and cognitive processes. SIGNIFICANCE STATEMENT While the neocortex is responsible for coordination of complex behavior, it requires communication with subcortical regions to do so. It is specifically cortical layer 5 (L5) that is thought to underlie these behaviors, although it is unknown whether this holds true across functionally different cortical areas. Using a selective viral tracing method and transgenic mice, we examined the connectivity of four cortical regions (somatosensory, visual, motor and prefrontal cortex) to assess the generalizability of these L5 projections. All areas of cortex projected to overlapping as well as distinct thalamic and brainstem structures. Terminals within these regions varied in size, implicating that L5 has a broad and diverse impact on behavior., (Copyright © 2020 the authors.)

Background: The relationship between poor oral health conditions and cognitive decline is unclear., Objective: To examine the association between oral health and cognition in humans and rats., Methods: In humans: a cross-sectional study was conducted. Cognitive levels were evaluated by the Mini Mental State Examination (MMSE); oral conditions were reflected by the number of missing index teeth, bleeding on probing, and probing pocket depth (PD). In rats: a ligature-induced (Lig) periodontitis model and Aβ25-35-induced model of Alzheimer's disease (AD) were established; tumor necrosis factor-α (TNF-α), interleukin 1 (IL-1), interleukin 6 (IL-6), and C-reactive protein levels in the hippocampus and cerebral cortex were detected., Results: MMSE scores for the number of missing index teeth ≥ 7 group were significantly lower than those in the ≤ 6 group. A negative relationship (correlation coefficient ρ = -0.310, P = 0.002) was observed between MMSE scores and number of missing index teeth. More missing index teeth and lower education levels were independent risk factors for cognitive decline. A negative relationship (correlation coefficient ρ = -0.214, P = 0.031) was observed between MMSE scores and average PD. TNF-α and IL-6 levels in the hippocampus of the Lig+AD group were significantly higher than those of the AD group. IL-1 and IL-6 levels in the cerebral cortex of the Lig+AD group were significantly higher than those of the AD group., Conclusion: Poor oral health conditions including more missing index teeth and higher average PD may be risk factors for cognitive decline. Periodontitis may increase inflammatory cytokines in rat models of AD., Competing Interests: The authors have declared that no competing interests exist.

We developed entangled link-augmented stretchable tissue-hydrogel (ELAST), a technology that transforms tissues into elastic hydrogels to enhance macromolecular accessibility and mechanical stability simultaneously. ELASTicized tissues are highly stretchable and compressible, which enables reversible shape transformation and faster delivery of probes into intact tissue specimens via mechanical thinning. This universal platform may facilitate rapid and scalable molecular phenotyping of large-scale biological systems, such as human organs.

Background: Glycerophospholipids were the main components of cerebral cortex lipids, and there was a close association between lipid homeostasis and human health. It has been reported that dietary DHA-enriched phosphatidylcholine (DHA-PC) and phosphatidylserine (DHA-PS) could improve brain function. However, it was unclear that whether supplementation of DHA-PC and DHA-PS could change lipid profiles in the brain of dementia animals., Methods: SAMP8 mice was fed with different diet patterns for 2 months, including high-fat diet and low-fat diet. After intervention with DHA-PC and DHA-PS for another 2 months, the lipid profile in cerebral cortex was determined by lipidomics in dementia mice., Results: High-fat diet could significantly decrease the levels of DHA-containing PS/pPE, DPA-containing PS, and AA-containing PE, which might exhibit the potential of lipid biomarkers for the prevention and diagnosis of AD. Notably, DHA-PC and DHA-PS remarkably recovered the lipid homeostasis in dementia mice. These might provide a potential novel therapy strategy and direction of dietary intervention for patients with cognitive decline., Conclusions: DHA-PC and DHA-PS could recover the content of brain DHA-containing PS and pPE in SAMP8 mice fed with high-fat diet.

The physiological actions of orally ingested peptides on the brain remain poorly understood. This study examined the effects of 39 orally administered synthetic Tyr-containing dipeptides on the enhancement of brain norepinephrine metabolism in mice by comparing the concentration of 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG). Although Tyr-Tyr administration increased blood and cerebral cortex (Cx) Tyr concentrations the most, Tyr-Trp increased Cx MHPG concentration the most. The oral administration of Tyr-Trp ameliorated a short-term memory deficit of a mouse model of cognitive dysfunction induced by amyloid beta peptide 25-35. Gene expression profiling of mouse brain using a microarray indicated that Tyr-Trp administration led to a wide variety of changes in mRNA levels, including the upregulation of genes encoding molecules involved in catecholamine metabolism. A comparative metabolome analysis of the Cx of mice given Tyr-Trp or Tyr-Tyr demonstrated that Tyr-Trp administration yielded higher concentrations of Trp and kynurenine pathway metabolites than Tyr-Tyr administration, as well as higher L-dopa levels, which is the initial product of catecholamine metabolism. Catecholamines were not significantly increased in the Cx of the Tyr-Tyr group compared with the Tyr-Trp group, despite a marked increase in Tyr. Presumably, Tyr-Trp administration enhances catecholamine synthesis and metabolism via the upregulation of genes involved in Tyr and Trp metabolism as well as metabolites of Tyr and Trp. These findings strongly suggest that orally ingested Tyr-Trp modulates the brain metabolome involved in catecholamine metabolism and contributes to higher brain function., Competing Interests: All authors except one author [H.M.] have no conflict of interest. H.M. is an employee of Nihon Bioresearch Inc. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The exquisite intricacies of neural circuits are fundamental to an animal's diverse and complex repertoire of sensory and motor functions. The ability to precisely map neural circuits and to selectively manipulate neural activity is critical to understanding brain function and has, therefore been a long-standing goal for neuroscientists. The recent development of optogenetic tools, combined with transgenic mouse lines, has endowed us with unprecedented spatiotemporal precision in circuit analysis. These advances greatly expand the scope of tractable experimental investigations. Here, in the first half of the review, we will present applications of optogenetics in identifying connectivity between different local neuronal cell types and of long-range projections with both in vitro and in vivo methods. We will then discuss how these tools can be used to reveal the functional roles of these cell-type specific connections in governing sensory information processing, and learning and memory in the visual cortex, somatosensory cortex, and motor cortex. Finally, we will discuss the prospect of new optogenetic tools and how their application can further advance modern neuroscience. In summary, this review serves as a primer to exemplify how optogenetics can be used in sophisticated modern circuit analyses at the levels of synapses, cells, network connectivity and behaviors., (Copyright © 2020 Lee, Lavoie, Liu, Chen and Liu.)

Inhibitory interneurons can evoke vasodilation and vasoconstriction, making them potential cellular drivers of neurovascular coupling. However, the specific regulatory roles played by particular interneuron subpopulations remain unclear. Our purpose was therefore to adopt a cell-specific optogenetic approach to investigate how somatostatin (SST) and neuronal nitric oxide synthase (nNOS)-expressing interneurons might influence the neurovascular relationship. In mice, specific activation of SST- or nNOS-interneurons was sufficient to evoke hemodynamic changes. In the case of nNOS-interneurons, robust hemodynamic changes occurred with minimal changes in neural activity, suggesting that the ability of blood oxygen level dependent functional magnetic resonance imaging (BOLD fMRI) to reliably reflect changes in neuronal activity may be dependent on type of neuron recruited. Conversely, activation of SST-interneurons produced robust changes in evoked neural activity with shallow cortical excitation and pronounced deep layer cortical inhibition. Prolonged activation of SST-interneurons often resulted in an increase in blood volume in the centrally activated area with an accompanying decrease in blood volume in the surrounding brain regions, analogous to the negative BOLD signal. These results demonstrate the role of specific populations of cortical interneurons in the active control of neurovascular function., (© The Author(s) 2019. Published by Oxford University Press.)

Domino cyclization reactions of N -aryl-1,4- and 1,5-benzoxazepine derivatives involving [1,5]-hydride shift or C(sp 2 )-H functionalization were investigated. Neuroprotective and acetylcholinesterase activities of the products were studied. Domino Knoevenagel-[1,5]-hydride shift-cyclization reaction of N -aryl-1,4-benzoxazepine derivatives with 1,3-dicarbonyl reagents having active methylene group afforded the 1,2,8,9-tetrahydro-7b H -quinolino [1,2- d ][1,4]benzoxazepine scaffold with different substitution pattern. The C(sp 3 )-H activation step of the tertiary amine moiety occurred with complete regioselectivity and the 6- endo cyclization took place in a complete diastereoselective manner. In two cases, the enantiomers of the chiral condensed new 1,4-benzoxazepine systems were separated by chiral HPLC, HPLC-ECD spectra were recorded, and absolute configurations were determined by time-dependent density functional theory- electronic circular dichroism (TDDFT-ECD) calculations. In contrast, the analogue reaction of the regioisomeric N -aryl-1,5-benzoxazepine derivative did not follow the above mechanism but instead the Knoevenagel intermediate reacted in an S E Ar reaction [C(sp 2 )-H functionalization] resulting in a condensed acridane derivative. The AChE inhibitory assays of the new derivatives revealed that the acridane derivative had a 6.98 μM IC 50 value.

Compulsive eating characterizes many binge-related eating disorders, yet its neurobiological basis is poorly understood. The insular cortex subserves visceral-emotional functions, including taste processing, and is implicated in drug craving and relapse. Here, via optoinhibition, we implicate projections from the anterior insular cortex to the nucleus accumbens as modulating highly compulsive-like food self-administration behaviors that result from intermittent access to a palatable, high-sucrose diet. We identified compulsive-like eating behavior in female rats through progressive ratio schedule self-administration and punishment-resistant responding, food reward tolerance and escalation of intake through 24-h energy intake and fixed-ratio operant self-administration sessions, and withdrawal-like irritability through the bottle brush test. We also identified an endocrine profile of heightened GLP-1 and PP but lower ghrelin that differentiated rats with the most compulsive-like eating behavior. Measures of compulsive eating severity also directly correlated to leptin, body weight and adiposity. Collectively, this novel model of compulsive-like eating symptoms demonstrates adaptations in insula-ventral striatal circuitry and metabolic regulatory hormones that warrant further study.

The ketoglutarate dehydrogenase complex (KGDHC) consists of three different subunits encoded by OGDH (or OGDHL), DLST, and DLD, combined in different stoichiometries. DLD subunit is shared between KGDHC and pyruvate dehydrogenase complex, branched-chain alpha-keto acid dehydrogenase complex, and the glycine cleavage system. Despite KGDHC's implication in neurodegenerative diseases, cell-specific localization of its subunits in the adult human brain has never been investigated. Here, we show that immunoreactivity of all known isoforms of OGDHL, OGDH, and DLST was detected exclusively in neurons of surgical human cortical tissue samples identified by their morphology and visualized by double labeling with fluorescent Nissl, while being absent from glia expressing GFAP, Aldhl1, myelin basic protein, Olig2, or IBA1. In contrast, DLD immunoreactivity was evident in both neurons and glia. Specificity of anti-KGDHC subunits antisera was verified by a decrease in staining of siRNA-treated human cancer cell lines directed against the respective coding gene products; furthermore, immunoreactivity of KGDHC subunits in human fibroblasts co-localized > 99% with mitotracker orange, while western blotting of 63 post-mortem brain samples and purified recombinant proteins afforded further assurance regarding antisera monospecificity. KGDHC subunit immunoreactivity correlated with data from the Human Protein Atlas as well as RNA-Seq data from the Allen Brain Atlas corresponding to genes coding for KGDHC components. Protein lysine succinylation, however, was immunohistochemically evident in all cortical cells; this was unexpected, because this posttranslational modification requires succinyl-CoA, the product of KGDHC. In view of the fact that glia of the human brain cortex lack succinate-CoA ligase, an enzyme producing succinyl-CoA when operating in reverse, protein lysine succinylation in these cells must exclusively rely on propionate and/or ketone body metabolism or some other yet to be discovered pathway encompassing succinyl-CoA.

Electrical kindling, repeated brain stimulation eventually resulting in seizures, is widely used as an animal model of epileptogenesis and epilepsy. However, the stimulation electrode used for electric kindling targets unknown neuronal populations and may introduce tissue damage and inflammation. Optogenetics can be used to circumvent these shortcomings by permitting millisecond control of activity in genetically defined neurons without gross injury or inflammation. Here we describe an easy step-by-step protocol for optogenetic kindling - optokindling - by which seizures are eventually elicited in initially healthy mice through repeated light stimulation of neurons expressing Channelrhodopsin-2 (ChR2). Chronic EEG recordings may be performed over large time scales to monitor activity while video camera monitoring may be used to assess the behavioral severity of seizures. In conclusion, with optokindling, neuroscientists can elucidate the circuit changes that underpin epilepsy while minimizing the contribution of confounding factors such as brain damage and inflammation., (Copyright © 2020 Cela and Sjöström.)

Background: Vitamin K (VK) exists in the form of phylloquinone (PK) and menaquinones (MKs). Roles of VK on cognitive health in the elderly are emerging, but there is limited evidence on VK uptake and metabolism in human brain., Objectives: The primary objective of this study was to characterize VK distribution in brains of an elderly population with varied cognitive function. In addition, associations among circulating (a biomarker of VK intake) and cerebral VK concentrations and cognition were investigated., Methods: Serum or plasma (n = 27) and brain samples from the frontal cortex (FC; n = 46) and the temporal cortex (TC; n = 33) were acquired from 48 decedents (aged 98-107 y; 25 demented and 23 nondemented) enrolled in the Georgia Centenarian Study. Both circulating and brain VK concentrations were measured using HPLC with fluorescence detection. Cognitive assessment was performed within 1 y prior to mortality. Partial correlations between serum/plasma or cerebral VK concentrations and cognitive function were performed, adjusting for covariates and separating by dementia and antithrombotic use., Results: MK-4 was the predominant vitamer in both FC (mean ± SD = 4.92 ± 2.31 pmol/g, ≥89.15% ± 5.09% of total VK) and TC (4.60 ± 2.11 pmol/g, ≥89.71% ± 4.43% of total VK) regardless of cognitive status. Antithrombotic users had 34.0% and 53.9% lower MK-4 concentrations in FC (P < 0.05) and TC (P < 0.001), respectively. Circulating PK was not correlated with cerebral MK-4 or total VK concentrations. Circulating PK concentrations were significantly associated with a wide range of cognitive measures in nondemented centenarians (P < 0.05). In contrast, cerebral MK-4 concentrations were not associated with cognitive performance, either before or after exclusion of antithrombotic users., Conclusions: Circulating VK concentrations are not related to cerebral MK-4 concentrations in centenarians. Cerebral MK-4 concentrations are tightly regulated over a range of VK intakes and cognitive function. Circulating PK may reflect intake of VK-rich foods containing other dietary components beneficial to cognitive health. Further investigation of VK uptake and metabolism in the brain is warranted., (Copyright © American Society for Nutrition 2019.)

In adult rats, omega-6 linoleic acid (LA, 18:2n-6) serves as a precursor to oxidized LA metabolites (OXLAMs) known to regulate multiple signaling processes in the brain. However, little is known regarding the levels or role(s) of LA and its metabolites during brain development. To address this gap, fatty acids within various brain lipid pools, and their oxidized metabolites (oxylipins) were quantified in brains from 1-day-old male and female pups using gas chromatography and liquid chromatography coupled to tandem mass spectrometry, respectively. Primary neuron-glia co-cultures derived from postnatal day 0-1 male and female rat neocortex were exposed to vehicle (0.1% ethanol), LA, the OXLAM 13-hydroxyoctadecadienoic acid (13-HODE), or prostaglandin E2 at 10-1000 nM for 48 h to test their effects on neuronal morphology. In both male and female pups, LA accounted for 1-3% of fatty acids detected in brain phospholipids and cholesteryl esters. It was not detected in triacylglycerols, and free fatty acids. Unesterified OXLAMs constituted 47-53% of measured unesterified oxylipins in males and females (vs. ~5-7% reported in adult rat brain). Of these, 13-HODE was the most abundant, accounting for 30-33% of measured OXLAMs. Brain fatty acid and OXLAM concentrations did not differ between sexes. LA and 13-HODE significantly increased axonal outgrowth. Separate analyses of cultures derived from male versus female pups revealed that LA at 1, 50, and 1000 nM, significantly increased axonal outgrowth in female but not male cortical neurons, whereas 13-HODE at 100 nM significantly increased axonal outgrowth in male but not female cortical neurons. prostaglandin E2 did not alter neuronal outgrowth in either sex. This study demonstrates that OXLAMs constitute the majority of unesterified oxylipins in the developing rat brain despite low relative abundance of their LA precursor, and highlights a novel role of LA and 13-HODE in differentially influencing neuronal morphogenesis in the developing male and female brain., (© 2019 International Society for Neurochemistry.)

Corticospinal neurons (CSNs) undertake direct cortical outputs to the spinal cord and innervate the upper limb through the brachial plexus. Our previous study has shown that the contralateral middle trunk transfer to the paralyzed upper extremity due to cerebral injury can reconstruct the functional cerebral cortex and improve the function of the paralyzed upper extremity. To interpret the cortical reconstruction and the motor improvement after the middle trunk transfer, we explored the distribution of CSNs connecting to the middle, upper, and lower trunk of the brachial plexus by retrograde trans-neuronal tracing using pseudorabies virus (PRV-EGFP or PRV-mRFP). We show that, rather than an individual specific area, these CSNs labelled by each trunk of the brachial plexus were widespread and mainly assembled within the primary motor cortex (M1), secondary motor cortex (M2), primary somatosensory cortex (S1), and slightly within the secondary somatosensory cortex (S2). The three trunk-labelled CSNs were intermingled in these cortices, and mostly connected to more than two trunks, especially the middle trunk-labelled CSNs with higher proportion of co-labelled neurons. Our findings revealed the distribution features of CSNs connecting to the adjacent spinal nerves that innervate the upper limb, which can improve our understanding of the corticospinal circuits associated with motor improvement and the functional cortical reconstruction after the middle trunk transfer., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)