A petavoxel fragment of human cerebral cortex reconstructed at nanoscale resolution.

To fully understand how the human brain works, knowledge of its structure at high resolution is needed. Presented here is a computationally intensive reconstruction of the ultrastructure of a cubic millimeter of human temporal cortex that was surgically removed to gain access to an underlying epileptic focus. It contains about 57,000 cells, about 230 millimeters of blood vessels, and about 150 million synapses and comprises 1.4 petabytes. Our analysis showed that glia outnumber neurons 2:1, oligodendrocytes were the most common cell, deep layer excitatory neurons could be classified on the basis of dendritic orientation, and among thousands of weak connections to each neuron, there exist rare powerful axonal inputs of up to 50 synapses. Further studies using this resource may bring valuable insights into the mysteries of the human brain.

Cortical gradients during naturalistic processing are hierarchical and modality-specific.

Understanding cortical topographic organization and how it supports complex perceptual and cognitive processes is a fundamental question in neuroscience. Previous work has characterized functional gradients that demonstrate large-scale principles of cortical organization. How these gradients are modulated by rich ecological stimuli remains unknown. Here, we utilize naturalistic stimuli via movie-fMRI to assess macroscale functional organization. We identify principal movie gradients that delineate separate hierarchies anchored in sensorimotor, visual, and auditory/language areas. At the opposite/heteromodal end of these perception-to-cognition axes, we find a more central role for the frontoparietal network along with the default network. Even across different movie stimuli, movie gradients demonstrated good reliability, suggesting that these hierarchies reflect a brain state common across different naturalistic conditions. The relative position of brain areas within movie gradients showed stronger and more numerous correlations with cognitive behavioral scores compared to resting state gradients. Together, these findings provide an ecologically valid representation of the principles underlying cortical organization while the brain is active and engaged in multimodal, dynamic perceptual and cognitive processing.

Layer 5 of cortex innervates the thalamic reticular nucleus in mice.

Neurons in the thalamic reticular nucleus (TRN) are a primary source of inhibition to the dorsal thalamus and, as they are innervated in part by the cortex, are a means of corticothalamic regulation. Previously, cortical inputs to the TRN were thought to originate solely from layer 6 (L6), but we recently reported the presence of putative synaptic terminals from layer 5 (L5) neurons in multiple cortical areas in the TRN [J. A. Prasad, B. J. Carroll, S. M. Sherman, J. Neurosci. 40, 5785-5796 (2020)]. Here, we demonstrate with electron microscopy that L5 terminals from multiple cortical regions make bona fide synapses in the TRN. We further use light microscopy to localize these synapses relative to recently described TRN subdivisions and show that L5 terminals target the edges of the somatosensory TRN, where neurons reciprocally connect to higher-order thalamus, and that L5 terminals are scarce in the core of the TRN, where neurons reciprocally connect to first-order thalamus. In contrast, L6 terminals densely innervate both edge and core subregions and are smaller than those from L5. These data suggest that a sparse but potent input from L5 neurons of multiple cortical regions to the TRN may yield transreticular inhibition targeted to higher-order thalamus.

Whole-brain neuronal MCT2 lactate transporter expression links metabolism to human brain structure and function.

Brain activity is constrained by local availability of chemical energy, which is generated through compartmentalized metabolic processes. By analyzing data of whole human brain gene expression, we characterize the spatial distribution of seven glucose and monocarboxylate membrane transporters that mediate astrocyte-neuron lactate shuttle transfer of energy. We found that the gene coding for neuronal MCT2 is the only gene enriched in cerebral cortex where its abundance is inversely correlated with cortical thickness. Coexpression network analysis revealed that MCT2 was the only gene participating in an organized gene cluster enriched in K[Formula: see text] dynamics. Indeed, the expression of K[Formula: see text] subunits, which mediate lactate increases with spiking activity, is spatially coupled to MCT2 distribution. Notably, MCT2 expression correlated with fluorodeoxyglucose positron emission tomography task-dependent glucose utilization. Finally, the MCT2 messenger RNA gradient closely overlaps with functional MRI brain regions associated with attention, arousal, and stress. Our results highlight neuronal MCT2 lactate transporter as a key component of the cross-talk between astrocytes and neurons and a link between metabolism, cortical structure, and state-dependent brain function.

Connectomic comparison of mouse and human cortex.

The human cerebral cortex houses 1000 times more neurons than that of the cerebral cortex of a mouse, but the possible differences in synaptic circuits between these species are still poorly understood. We used three-dimensional electron microscopy of mouse, macaque, and human cortical samples to study their cell type composition and synaptic circuit architecture. The 2.5-fold increase in interneurons in humans compared with mice was compensated by a change in axonal connection probabilities and therefore did not yield a commensurate increase in inhibitory-versus-excitatory synaptic input balance on human pyramidal cells. Rather, increased inhibition created an expanded interneuron-to-interneuron network, driven by an expansion of interneuron-targeting interneuron types and an increase in their synaptic selectivity for interneuron innervation. These constitute key neuronal network alterations in the human cortex.

Thalamocortical axons regulate neurogenesis and laminar fates in the early sensory cortex.

Area-specific axonal projections from the mammalian thalamus shape unique cellular organization in target areas in the adult neocortex. How these axons control neurogenesis and early neuronal fate specification is poorly understood. By using mutant mice lacking the majority of thalamocortical axons, we show that these axons are required for the production and specification of the proper number of layer 4 neurons in primary sensory areas by the neonatal stage. Part of these area-specific roles is played by the thalamus-derived molecule, VGF. Our work reveals that extrinsic cues from sensory thalamic projections have an early role in the formation of cortical cytoarchitecture by enhancing the production and specification of layer 4 neurons.

Aluminum impairs cognitive function by activating DDX3X-NLRP3-mediated pyroptosis signaling pathway.

INTRODUCTION: Aluminum is a kind of chemical contaminants in food which can induce neurotoxicity. Aluminum exposure is closely related to neurodegenerative diseases (ND), in which neuroinflammation might involve. However, the molecular mechanism of aluminum-induced neuroinflammation through pyroptosis is not fully clarified yet. MATERIAL AND METHODS: The mice model of subacute exposure to aluminum chloride (AlCl3) was established. BV2 microglia cells was treated with AlCl3 in vitro. Resveratrol (Rsv) was adopted as intervention agent. RESULTS: Our results showed that aluminum induced cognitive impairment, destroying blood brain barrier (BBB), and causing nerve injury in mice. Meanwhile, aluminum could stimulate nucleotide oligomerization domain-like receptor family pyrin domain containing protein 3 (NLRP3) inflammasome assembly and activate caspase-1 (CASP1), inducing gasdermin D (GSDMD)-mediated pyroptosis signaling, releasing cytokines IL-1ß and IL-18, further promoting the activation of glial cells to magnify neuroinflammatory response. Moreover, DEAD-box helicase 3 X-linked (DDX3X) and stress granule RasGAP SH3-domain-binding protein 1 (G3BP1) both participated in neuroinflammation induced by aluminum. When co-treated with Rsv, these injuries were alleviated to some extent. CONCLUSION: Aluminum exposure could induce nerve cell pyroptosis and neuroinflammation by DDX3X-NLRP3 inflammasome signaling pathway, which could be rescued via Rsv activating sirtuin 1 (SIRT1).

PUPIL enables mapping and stamping of transient electrical connectivity in developing nervous systems.

Currently, many genetic methods are available for mapping chemical connectivity, but analogous methods for electrical synapses are lacking. Here, we present pupylation-based interaction labeling (PUPIL), a genetically encoded system for noninvasively mapping and stamping transient electrical synapses in the mouse brain. Upon fusion of connexin 26 (CX26) with the ligase PafA, pupylation yields tag puncta following conjugation of its substrate, a biotin- or fluorescent-protein-tagged PupE, to the neighboring proteins of electrical synapses containing CX26-PafA. Tag puncta are validated to correlate well with functional electrical synapses in immature neurons. Furthermore, puncta are retained in mature neurons when electrical synapses mostly disappear-suggesting successful stamping. We use PUPIL to uncover spatial subcellular localizations of electrical synapses and approach their physiological functions during development. Thus, PUPIL is a powerful tool for probing electrical connectivity patterns in complex nervous systems and has great potential for transient receptors and ion channels as well.

Super-resolution study of PIAS SUMO E3-ligases in hippocampal and cortical neurons.

The SUMOylation machinery is a regulator of neuronal activity and synaptic plasticity. It is composed of SUMO isoforms and specialized enzymes named E1, E2 and E3 SUMO ligases. Recent studies have highlighted how SUMO isoforms and E2 enzymes localize with synaptic markers to support previous functional studies but less information is available on E3 ligases. PIAS proteins - belonging to the protein inhibitor of activated STAT (PIAS) SUMO E3-ligase family - are the best-characterized SUMO E3-ligases and have been linked to the formation of spatial memory in rodents. Whether however they exert their function co-localizing with synaptic markers is still unclear. In this study, we applied for the first time structured illumination microscopy (SIM) to PIAS ligases to investigate the co-localization of PIAS1 and PIAS3 with synaptic markers in hippocampal and cortical murine neurons. The results indicate partial co-localization of PIAS1 and PIAS3 with synaptic markers in hippocampal neurons and much rarer occurrence in cortical neurons. This is in line with previous super-resolution reports describing the co-localization with synaptic markers of other components of the SUMOylation machinery.

1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) Urea Exerts Neuro-Protective Effects Against Ischemic Injury via Suppressing JNK/p38 MAPK-Mediated Mitochondrial Apoptosis Pathway.

BACKGROUND: 1-trifluoromethoxyphenyl-3-(1- propionylpiperidin-4-yl) urea (TPPU) is a novel soluble epoxide hydrolase inhibitor which can protect against cerebral ischemic injury in middle cerebral artery occlusion rat model. However, the effects and potential mechanisms of TPPU on mitochondrial dysfunction are poorly understood. MATERIALS AND METHODS: In oxygen-glucose deprivation/reperfusion (OGD/R)-induced cortical neurons, the effect of TPPU on cell viability was measured by MTT assay and apoptosis was evaluated using TUNEL assay. Mitochondria were observed by transmission electron microscopy and Mitotracker green staining assay, mitochondrial membrane potential was determined by JC-1 staining assay, activities of mitochondrial respiratory chain complexes (MRCC) I-IV and ATPase were measured by MRCC Activity Assay Kits and spectrophotometer. Western blot was used to investigate the effects of TPPU on apoptosis-related proteins. RESULTS: TPPU treatment demonstrated significant protective effect on the OGD/R-induced cortical neurons by reducing cell death and number of apoptotic cells, stabilizing mitochondrial ultrastructure and morphology, increasing mitochondrial membrane potential and activities of MRCC I-IV and ATPase. Furthermore, TPPU treatment might effectively reverse the upregulation of caspase-3, Bax, p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal protein kinase (JNK), alleviate the inhibition of Bcl-2 in OGD/R-induced cortical neurons. CONCLUSIONS: TPPU exerts a marked neuroprotective effect against mitochondrial dysfunction after cerebral ischemia potentially via suppressing JNK/p38 MAPK-mediated mitochondrial apoptosis signal pathway, it may be a promising neuroprotective agent for cerebral ischemia.

Olig2 knockdown alleviates hypoxic-ischemic brain damage in newborn rats.

OBJECTIVES: Neuronal damage is an important pathological mechanism in neonatal hypoxic-ischemic brain damage (HIBD). We found in our previous studies that oligodendrocyte transcription factor 2 (Olig2) downregulation was able to increase cell survival in the brain. However, the specific mechanism has yet to be clarified. METHODS: Sprague-Dawley rats aged 3 d were randomly divided into three groups: the normal control group, the Olig2-RNAi group, and the RNAi-negative control group. The normal control group received no treatment, the Olig2-RNAi group received the Olig2 RNAi adenovirus, and the RNAi-negative control group was given the control adenovirus after the completion of the HIBD model. Infarct lesions and their volumes were observed by triphenyltetrazolium chloride (TTC) staining 3 d after the completion of the adenovirus local injection. The condition of the tissue was characterized by hematoxylin-eosin staining 7 d after the model was established, and cell viability was determined by azure methylene blue staining. Subcellular damage was analyzed by transmission electron microscopy. Rotarod analysis was performed to detect moving behavior ability and an MWM assay was conducted to evaluate the memory. RESULTS: TTC staining showed a smaller brain injury area in the Olig2-RNAi group than in the RNAi-negative control group. Hematoxylin-eosin staining indicated the presence of severe cell injury in the hippocampal region after HIBD, which improved after Olig2 knockdown. Azure methylene blue staining and electron microscopy results suggested that the cells improved after Olig2 knockdown. The rats stayed longer on the rotating rod, and their latency in the water maze test was gradually shortened relative to that of the rats in the Olig2-RNAi negative control group. CONCLUSION: Olig2 knockdown can promote the repair of hypoxic-ischemic brain damage in newborn rats.

Topological reorganizations of mitochondria isolated from rat brain after 72 hours of paradoxical sleep deprivation, revealed by electron cryo-tomography.

Sleep deprivation has profound influence on several aspects of health and disease. Mitochondria dysfunction has been implicated to play an essential role in the neuronal cellular damage induced by sleep deprivation, but little is known about how neuronal mitochondrial ultrastructure is affected under sleep deprivation. In this report, we utilized electron cryo-tomography to reconstruct the three-dimensional (3-D) mitochondrial structure and extracted morphometric parameters to quantitatively characterize its reorganizations. Isolated mitochondria from the hippocampus and cerebral cortex of adult male Sprague-Dawley rats after 72 h of paradoxical sleep deprivation (PSD) were reconstructed and analyzed. Statistical analysis of six morphometric parameters specific to the mitochondrial inner membrane topology revealed identical pattern of changes in both the hippocampus and cerebral cortex but with higher significance levels in the hippocampus. The structural differences were indistinguishable by conventional phenotypic methods based on two-dimensional electron microscopy images or 3-D electron tomography reconstructions. Furthermore, to correlate structure alterations with mitochondrial functions, high-resolution respirometry was employed to investigate the effects of PSD on mitochondrial respiration, which showed that PSD significantly suppressed the mitochondrial respiratory capacity of the hippocampus, whereas the isolated mitochondria from the cerebral cortex were less affected. These results demonstrate the capability of the morphometric parameters for quantifying complex structural reorganizations and suggest a correlation between PSD and inner membrane architecture/respiratory functions of the brain mitochondria with variable effects in different brain regions.

The Rab7 effector WDR91 promotes autophagy-lysosome degradation in neurons by regulating lysosome fusion.

The effectors of the Rab7 small GTPase play multiple roles in Rab7-dependent endosome-lysosome and autophagy-lysosome pathways. However, it is largely unknown how distinct Rab7 effectors coordinate to maintain the homeostasis of late endosomes and lysosomes to ensure appropriate endolysosomal and autolysosomal degradation. Here we report that WDR91, a Rab7 effector required for early-to-late endosome conversion, is essential for lysosome function and homeostasis. Mice lacking Wdr91 specifically in the central nervous system exhibited behavioral defects and marked neuronal loss in the cerebral and cerebellar cortices. At the cellular level, WDR91 deficiency causes PtdIns3P-independent enlargement and dysfunction of lysosomes, leading to accumulation of autophagic cargoes in mouse neurons. WDR91 competes with the VPS41 subunit of the HOPS complex, another Rab7 effector, for binding to Rab7, thereby facilitating Rab7-dependent lysosome fusion in a controlled manner. WDR91 thus maintains an appropriate level of lysosome fusion to guard the normal function and survival of neurons.

Environmental influences on the pace of brain development.

Childhood socio-economic status (SES), a measure of the availability of material and social resources, is one of the strongest predictors of lifelong well-being. Here we review evidence that experiences associated with childhood SES affect not only the outcome but also the pace of brain development. We argue that higher childhood SES is associated with protracted structural brain development and a prolonged trajectory of functional network segregation, ultimately leading to more efficient cortical networks in adulthood. We hypothesize that greater exposure to chronic stress accelerates brain maturation, whereas greater access to novel positive experiences decelerates maturation. We discuss the impact of variation in the pace of brain development on plasticity and learning. We provide a generative theoretical framework to catalyse future basic science and translational research on environmental influences on brain development.

Morphological Study of the Cortical and Thalamic Glutamatergic Synaptic Inputs of Striatal Parvalbumin Interneurons in Rats.

Parvalbumin-immunoreactive (Parv+) interneurons is an important component of striatal GABAergic microcircuits, which receive excitatory inputs from the cortex and thalamus, and then target striatal projection neurons. The present study aimed to examine ultrastructural synaptic connection features of Parv+ neruons with cortical and thalamic input, and striatal projection neurons by using immuno-electron microscopy (immuno-EM) and immunofluorescence techniques. Our results showed that both Parv+ somas and dendrites received numerous asymmetric synaptic inputs, and Parv+ terminals formed symmetric synapses with Parv- somas, dendrites and spine bases. Most interestingly, spine bases targeted by Parv+ terminals simultaneously received excitatory inputs at their heads. Electrical stimulation of the motor cortex (M1) induced higher proportion of striatal Parv+ neurons express c-Jun than stimulation of the parafascicular nucleus (PFN), and indicated that cortical- and thalamic-inputs differentially modulate Parv+ neurons. Consistent with that, both Parv + soma and dendrites received more VGlut1+ than VGlut2+ terminals. However, the proportion of VGlut1+ terminal targeting onto Parv+ proximal and distal dendrites was not different, but VGlut2+ terminals tended to target Parv+ somas and proximal dendrites than distal dendrites. These functional and morphological results suggested excitatory cortical and thalamic glutamatergic inputs differently modulate Parv+ interneurons, which provided inhibition inputs onto striatal projection neurons. To maintain the balance between the cortex and thalamus onto Parv+ interneurons may be an important therapeutic target for neurological disorders.

Alpha-Lipoic Acid Attenuates Cadmium- and Lead-Induced Neurotoxicity by Inhibiting Both Endoplasmic-Reticulum Stress and Activation of Fas/FasL and Mitochondrial Apoptotic Pathways in Rat Cerebral Cortex.

Although many studies have reported toxic effects of cadmium (Cd) and lead (Pb) in the central nervous system, few studies have investigated the combined toxicity of Cd and Pb. The mechanisms by which these combined heavy metals induce toxicity, as well as effective means to exert neuroprotection from these agents, remain poorly understood. To investigate the protective effects of alpha-lipoic acid (α-LA) on Cd- and/or Pb-induced cortical damage in rats, 48 Sprague-Dawley rats were exposed to drinking water containing 50 mg/L of Cd and/or 300 mg/L of Pb for 12 weeks, in the presence or absence of α-LA co-treatment (50 mg/kg) via gavage. We observed that exposure to Cd and/or Pb decreased the brain weight/body weight ratio and increased Cd and/or Pb contents as well as ultrastructural damage to the cerebral cortex. Cd and/or Pb also induced endoplasmic-reticulum (ER) stress and activated Fas (CD95/APO-1)/Fas ligand (FasL) and mitochondrial apoptotic pathways. Furthermore, co-treatment of Cd and Pb further exacerbated part of these phenotypes than treatment of Cd or Pb alone. However, simultaneous supplementation with α-LA attenuated Cd and/or Pb-induced neurotoxicity by increasing the brain weight/body weight ratio, reducing Cd and/or Pb contents, ameliorating both nuclear/mitochondrial damage and ER stress, and attenuating activation of Fas/FasL and mitochondrial apoptotic pathways. Collectively, our results indicate that the accumulation of Cd and/or Pb causes cortical damage and that α-LA exerts protection against Cd- and/or Pb-induced neurotoxicity. These findings highlight that α-LA may be exploited for the treatment and prevention of Cd- and/or Pb-induced neurotoxicity.

Age-related ultrastructural neurovascular changes in the female mouse cortex and hippocampus.

Blood-brain barrier (BBB) breakdown occurs in aging and neurodegenerative diseases. Although age-associated alterations have previously been described, most studies focused in male brains; hence, little is known about BBB breakdown in females. This study measured ultrastructural features in the aging female BBB using transmission electron microscopy and 3-dimensional reconstruction of cortical and hippocampal capillaries from 6- and 24-month-old female C57BL/6J mice. Aged cortical capillaries showed more changes than hippocampal capillaries. Specifically, the aged cortex showed thicker basement membrane, higher number and volume of endothelial pseudopods, decreased endothelial mitochondrial number, larger pericyte mitochondria, higher pericyte-endothelial cell contact, and increased tight junction tortuosity compared with young animals. Only increased basement membrane thickness and pericyte mitochondrial volume were observed in the aged hippocampus. Regional comparison revealed significant differences in endothelial pseudopods and tight junctions between the cortex and hippocampus of 24-month-old mice. Therefore, the aging female BBB shows region-specific ultrastructural alterations that may lead to oxidative stress and abnormal capillary blood flow and barrier stability, potentially contributing to cerebrovascular diseases, particularly in postmenopausal women.

The natural axis of transmitter receptor distribution in the human cerebral cortex.

Transmitter receptors constitute a key component of the molecular machinery for intercellular communication in the brain. Recent efforts have mapped the density of diverse transmitter receptors across the human cerebral cortex with an unprecedented level of detail. Here, we distill these observations into key organizational principles. We demonstrate that receptor densities form a natural axis in the human cerebral cortex, reflecting decreases in differentiation at the level of laminar organization and a sensory-to-association axis at the functional level. Along this natural axis, key organizational principles are discerned: progressive molecular diversity (increase of the diversity of receptor density); excitation/inhibition (increase of the ratio of excitatory-to-inhibitory receptor density); and mirrored, orderly changes of the density of ionotropic and metabotropic receptors. The uncovered natural axis formed by the distribution of receptors aligns with the axis that is formed by other dimensions of cortical organization, such as the myelo- and cytoarchitectonic levels. Therefore, the uncovered natural axis constitutes a unifying organizational feature linking multiple dimensions of the cerebral cortex, thus bringing order to the heterogeneity of cortical organization.

FMRP regulates the subcellular distribution of cortical dendritic spine density in a non-cell-autonomous manner.

Fragile X syndrome (FXS) is the most common form of intellectual disability that arises from the dysfunction of a single gene-Fmr1. The main neuroanatomical correlate of FXS is elevated dendritic spine density on cortical pyramidal neurons, which has been modeled in Fmr1-/Y mice. However, the cell-autonomous contribution of Fmr1 on cortical dendritic spine density has not been assessed. Even less is known about the role of Fmr1 in heterozygous female mosaic mice, which are a putative model for human Fmr1 full mutation carriers (i.e., are heterozygous for the full Fmr1-silencing mutation). In this neuroanatomical study, spine density in cortical pyramidal neurons of Fmr1+/- and Fmr1-/Y mice was studied at multiple subcellular compartments, layers, and brain regions. Spine density in Fmr1+/- mice is higher than WT but lower than Fmr1-/Y. Not all subcellular compartments in layer V Fmr1+/- and Fmr1-/Y cortical pyramidal neurons are equally affected: the apical dendrite, a key subcellular compartment, is principally affected over basal dendrites. Within apical dendrites, spine density is differentially affected across branch orders. Finally, identification of FMRP-positive and FMRP-negative neurons within Fmr1+/- permitted the study of the cell-autonomous effect of Fmr1 on spine density. Surprisingly, layer V cortical pyramidal spine density between FMRP-positive and FMRP-negative neurons does not differ, suggesting that the regulation of the primary neuroanatomical defect of FXS-elevated spine density-is non-cell-autonomous.

Mapping the molecular and cellular complexity of cortical malformations.

The cerebral cortex is an intricate structure that controls human features such as language and cognition. Cortical functions rely on specialized neurons that emerge during development from complex molecular and cellular interactions. Neurodevelopmental disorders occur when one or several of these steps is incorrectly executed. Although a number of causal genes and disease phenotypes have been identified, the sequence of events linking molecular disruption to clinical expression mostly remains obscure. Here, focusing on human malformations of cortical development, we illustrate how complex interactions at the genetic, cellular, and circuit levels together contribute to diversity and variability in disease phenotypes. Using specific examples and an online resource, we propose that a multilevel assessment of disease processes is key to identifying points of vulnerability and developing new therapeutic strategies.