Optimisation and validation of hydrogel-based brain tissue clearing shows uniform expansion across anatomical regions and spatial scales

Imaging of fxed tissue is routine in experimental neuroscience, but is limited by the depth of tissue that can be imaged using conventional methods. Optical clearing of brain tissue using hydrogelbased methods (e.g. CLARITY) allows imaging of large volumes of tissue and is rapidly becoming commonplace in the feld. However, these methods sufer from a lack of standardized protocols and validation of the efect they have upon tissue morphology. We present a simple and reliable protocol for tissue clearing along with a quantitative assessment of the efect of tissue clearing upon morphology. Tissue clearing caused tissue swelling (compared to conventional methods), but this swelling was shown to be similar across spatial scales and the variation was within limits acceptable to the feld. The results of many studies rely upon an assumption of uniformity in tissue swelling, and by demonstrating this quantitatively, research using these methods can be interpreted more reliably

Male-Dominant Effects of Chd8 Haploinsufficiency on Synaptic Phenotypes during Development in Mouse Prefrontal Cortex

This is the final version. Available on open access from the American Chemical Society via the DOI in this recordCHD8 is a high penetrance, high confidence risk gene for autism spectrum disorder (ASD), a neurodevelopmental disorder that is substantially more prevalent among males than among females. Recent studies have demonstrated variable sex differences in the behaviors and synaptic phenotypes of mice carrying different heterozygous ASD-associated mutations in Chd8. We examined functional and structural cellular phenotypes linked to synaptic transmission in deep layer pyramidal neurons of the prefrontal cortex in male and female mice carrying a heterozygous, loss-of-function Chd8 mutation in the C57BL/6J strain across development from postnatal day 2 to adulthood. Notably, excitatory neurotransmission was decreased only in Chd8+/- males with no differences in Chd8+/- females, and the majority of alterations in inhibitory transmission were found in males. Similarly, analysis of cellular morphology showed male-specific effects of reduced Chd8 expression. Both functional and structural phenotypes were most prominent at postnatal days 14-20, a stage approximately corresponding to childhood. Our findings suggest that the effects of Chd8 mutation are predominantly seen in males and are maximal during childhood.Simons FoundationBiotechnology and Biological Sciences Research Council (BBSRC)KHP Challenge FundBrain & Behavior Research FoundationKing’s Bioscience InstituteSt Thomas’ CharityWellcome Trus

The intellectual disability risk gene Kdm5b regulates long term memory consolidation in the hippocampus

This is the author accepted manuscript. the final version is available from the Society for Neuroscience via the DOI in this recordData availability: RNAseq data (fastq files) were deposited at the Gene Expression Omnibus (GEO) archive under the accession number GSE240887 and made freely available upon publicationThe histone lysine demethylase KDM5B is implicated in recessive intellectual disability disorders and heterozygous, protein truncating variants in KDM5B are associated with reduced cognitive function in the population. The KDM5 family of lysine demethylases has developmental and homeostatic functions in the brain, some of which appear to be independent of lysine demethylase activity. To determine the functions of KDM5B in hippocampus-dependent learning and memory, we first studied male and female mice homozygous for a Kdm5b ΔARID allele that lacks demethylase activity. Kdm5b ΔARID/ΔARID mice exhibited hyperactivity and long-term memory deficits in hippocampus-dependent learning tasks. The expression of immediate early, activity-dependent genes was downregulated in these mice and hyperactivated upon learning stimulus compared to wildtype mice. A number of other learning-associated genes was also significantly dysregulated in the Kdm5b ΔARID/ΔARID hippocampus. Next, we knocked down Kdm5b specifically in the adult, wildtype mouse hippocampus with shRNA. Kdm5b knockdown resulted in spontaneous seizures, hyperactivity and hippocampus-dependent long-term memory and long-term potentiation deficits. These findings identify KDM5B as a critical regulator of gene expression and synaptic plasticity in the adult hippocampus and suggest that at least some of the cognitive phenotypes associated with KDM5B gene variants are caused by direct effects on memory consolidation mechanisms. Significance statement The histone lysine demethylase KDM5B has been implicated in cognitive performance and intellectual disability conditions in the human population. In the present manuscript we show that mice expressing a demethylase-deficient KDM5B and mice with a specific knockdown of KDM5B in the adult hippocampus exhibit hippocampus-dependent learning and memory phenotypes. Molecular analyses suggest a key role for KDM5B in regulating the dynamic expression of activity-regulated genes during memory consolidation. Deficits in LTP are present in mice with KDM5B knockdown. Together, these findings provide the first evidence for a direct function for KDM5B in memory consolidation in the hippocampus.Medical Research Council (MRC)National Institute of AgingWellcome Trus

The intellectual disability risk gene Kdm5b regulates long term memory consolidation in the hippocampus

The histone lysine demethylase KDM5B is implicated in recessive intellectual disability disorders and heterozygous, protein truncating variants in KDM5B are associated with reduced cognitive function in the population. The KDM5 family of lysine demethylases has developmental and homeostatic functions in the brain, some of which appear to be independent of lysine demethylase activity. To determine the functions of KDM5B in hippocampus-dependent learning and memory, we first studied male and female mice homozygous for a Kdm5bΔARID allele that lacks demethylase activity. Kdm5bΔARID/ΔARID mice exhibited hyperactivity and long-term memory deficits in hippocampus-dependent learning tasks. The expression of immediate early, activity-dependent genes was downregulated in these mice and hyperactivated upon learning stimulus compared to wildtype mice. A number of other learning-associated genes was also significantly dysregulated in the Kdm5bΔARID/ΔARID hippocampus. Next, we knocked down Kdm5b specifically in the adult, wildtype mouse hippocampus with shRNA. Kdm5b knockdown resulted in spontaneous seizures, hyperactivity and hippocampus-dependent long-term memory and long-term potentiation deficits. These findings identify KDM5B as a critical regulator of gene expression and synaptic plasticity in the adult hippocampus and suggest that at least some of the cognitive phenotypes associated with KDM5B gene variants are caused by direct effects on memory consolidation mechanisms. Significance statement The histone lysine demethylase KDM5B has been implicated in cognitive performance and intellectual disability conditions in the human population. In the present manuscript we show that mice expressing a demethylase-deficient KDM5B and mice with a specific knockdown of KDM5B in the adult hippocampus exhibit hippocampus-dependent learning and memory phenotypes. Molecular analyses suggest a key role for KDM5B in regulating the dynamic expression of activity-regulated genes during memory consolidation. Deficits in LTP are present in mice with KDM5B knockdown. Together, these findings provide the first evidence for a direct function for KDM5B in memory consolidation in the hippocampus

Africa and the global carbon cycle

The African continent has a large and growing role in the global carbon cycle, with potentially important climate change implications. However, the sparse observation network in and around the African continent means that Africa is one of the weakest links in our understanding of the global carbon cycle. Here, we combine data from regional and global inventories as well as forward and inverse model analyses to appraise what is known about Africa's continental-scale carbon dynamics. With low fossil emissions and productivity that largely compensates respiration, land conversion is Africa's primary net carbon release, much of it through burning of forests. Savanna fire emissions, though large, represent a short-term source that is offset by ensuing regrowth. While current data suggest a near zero decadal-scale carbon balance, interannual climate fluctuations (especially drought) induce sizeable variability in net ecosystem productivity and savanna fire emissions such that Africa is a major source of interannual variability in global atmospheric CO(2). Considering the continent's sizeable carbon stocks, their seemingly high vulnerability to anticipated climate and land use change, as well as growing populations and industrialization, Africa's carbon emissions and their interannual variability are likely to undergo substantial increases through the 21st century

SOX2 Co-Occupies Distal Enhancer Elements with Distinct POU Factors in ESCs and NPCs to Specify Cell State

SOX2 is a master regulator of both pluripotent embryonic stem cells (ESCs) and multipotent neural progenitor cells (NPCs); however, we currently lack a detailed understanding of how SOX2 controls these distinct stem cell populations. Here we show by genome-wide analysis that, while SOX2 bound to a distinct set of gene promoters in ESCs and NPCs, the majority of regions coincided with unique distal enhancer elements, important cis-acting regulators of tissue-specific gene expression programs. Notably, SOX2 bound the same consensus DNA motif in both cell types, suggesting that additional factors contribute to target specificity. We found that, similar to its association with OCT4 (Pou5f1) in ESCs, the related POU family member BRN2 (Pou3f2) co-occupied a large set of putative distal enhancers with SOX2 in NPCs. Forced expression of BRN2 in ESCs led to functional recruitment of SOX2 to a subset of NPC-specific targets and to precocious differentiation toward a neural-like state. Further analysis of the bound sequences revealed differences in the distances of SOX and POU peaks in the two cell types and identified motifs for additional transcription factors. Together, these data suggest that SOX2 controls a larger network of genes than previously anticipated through binding of distal enhancers and that transitions in POU partner factors may control tissue-specific transcriptional programs. Our findings have important implications for understanding lineage specification and somatic cell reprogramming, where SOX2, OCT4, and BRN2 have been shown to be key factors

Neuronal receptors display cytoskeleton-independent directed motion on the plasma membrane

Directed transport of transmembrane proteins is generally believed to occur via intracellular transport vesicles. However, using single-particle tracking in rat hippocampal neurons with a pH-sensitive quantum dot probe that specifically reports surface movement of receptors, we have identified a subpopulation of neuronal EphB2 receptors that exhibit directed motion between synapses within the plasma membrane itself. This receptor movement occurs independently of the cytoskeleton but is dependent on cholesterol and is regulated by neuronal activity

The within-subject application of diffusion tensor MRI and CLARITY reveals brain structural changes in Nrxn2 deletion mice

Background: Of the many genetic mutations known to increase the risk of autism spectrum disorder, a large proportion cluster upon synaptic proteins. One such family of presynaptic proteins are the neurexins (NRXN), and recent genetic and mouse evidence has suggested a causative role for NRXN2 in generating altered social behaviours. Autism has been conceptualised as a disorder of atypical connectivity, yet how single-gene mutations affect such connectivity remains under-explored. To attempt to address this, we have developed a quantitative analysis of microstructure and structural connectivity leveraging diffusion tensor MRI (DTI) with high-resolution 3D imaging in optically cleared (CLARITY) brain tissue in the same mouse, applied here to the Nrxn2α knockout (KO) model. Methods: Fixed brains of Nrxn2α KO mice underwent DTI using 9.4 T MRI, and diffusion properties of socially relevant brain regions were quantified. The same tissue was then subjected to CLARITY to immunolabel axons and cell bodies, which were also quantified. Results: DTI revealed increases in fractional anisotropy in the amygdala (including the basolateral nuclei), the anterior cingulate cortex, the orbitofrontal cortex and the hippocampus. Axial diffusivity of the anterior cingulate cortex and orbitofrontal cortex was significantly increased in Nrxn2α KO mice, as were tracts between the amygdala and the orbitofrontal cortex. Using CLARITY, we find significantly altered axonal orientation in the amygdala, orbitofrontal cortex and the anterior cingulate cortex, which was unrelated to cell density. Conclusions: Our findings demonstrate that deleting a single neurexin gene (Nrxn2α) induces atypical structural connectivity within socially relevant brain regions. More generally, our combined within-subject DTI and CLARITY approach presents a new, more sensitive method of revealing hitherto undetectable differences in the autistic brain