DISTINCT MOLECULAR AND MORPHOLOGICAL SUBCIRCUITS OF THE SUBPLATE NEURONS

Subplate neurons (SPNs) are a population of neurons in the mammalian cerebral cortex that exist predominantly in the prenatal and early postnatal period. Loss of SPNs prevents the functional maturation of the cerebral cortex. SPNs receive subcortical input from the thalamus and relay this information to the developing cortical plate and thereby can influence cortical activity in a feed-forward manner. Little is known about potential feedback projections from the cortical plate to SPN. SPNs are also a heterogeneous population in terms of molecular and morphological identity. And the functional role of the different subpopulation of SPN remains poorly defined. This is mainly due to the lack of tools- i.e. transgenic lines and reporters to target and manipulate the SPNs at different stages of development. Hence the functional significance of the molecular diversity remains unexplored. In this study, we used a combination of genetic, molecular, anatomical and physiological approaches to address these questions and also to identify and characterize transgenic `tools' to manipulate the SPN. We identified and characterized a set of reporters and transgenic lines expressing Cre recombinase or green fluorescent protein with different levels of specificity in the subplate (SP). Using these transgenic driver lines and specific antibodies, we find that defined SPNs project to the main thalamo-recipient layers - L4 and L1 - and the spatial pattern of SPN projections to layer 4 is related to the spatial pattern of thalamo-cortical projections. However different subclasses have distinct patterns of projections with respect to the thalamic afferents. While certain subclasses have been shown to project locally, we observe that certain cell types of SPN also extend long-range projections to different thalamic nuclei. Thus molecularly defined SPN cell types are differentially integrated into the thalamo-cortical and intra-cortical connectivity. We also find a laminar difference in intra-cortical connectivity of the SPN. The first class of SPNs receives inputs from only deep cortical layers, while the second class of SPNs receives inputs from deep as well as superficial layers including layer 4 and are located more superficially. These superficial cortical inputs to SPNs emerge in the second postnatal week. Taken together, we demonstrate the presence of distinct laminar and molecular circuits in the developing subplate and characterize yet another level of heterogeneity of this population

Subset of cortical layer 6b neurons selectively innervates higher order thalamic nuclei in mice

The thalamus receives input from 3 distinct cortical layers, but input from only 2 of these has been well characterized. We therefore investigated whether the third input, derived from layer 6b, is more similar to the projections from layer 6a or layer 5. We studied the projections of a restricted population of deep layer 6 cells (“layer 6b cells”) taking advantage of the transgenic mouse Tg(Drd1a-cre)FK164Gsat/Mmucd (Drd1a-Cre), that selectively expresses Cre-recombinase in a subpopulation of layer 6b neurons across the entire cortical mantle. At P8, 18% of layer 6b neurons are labeled with Drd1a-Cre::tdTomato in somatosensory cortex (SS), and some co-express known layer 6b markers. Using Cre-dependent viral tracing, we identified topographical projections to higher order thalamic nuclei. VGluT1+ synapses formed by labeled layer 6b projections were found in posterior thalamic nucleus (Po) but not in the (pre)thalamic reticular nucleus (TRN). The lack of TRN collaterals was confirmed with single-cell tracing from SS. Transmission electron microscopy comparison of terminal varicosities from layer 5 and layer 6b axons in Po showed that L6b varicosities are markedly smaller and simpler than the majority from L5. Our results suggest that L6b projections to the thalamus are distinct from both L5 and L6a projectionsZ.M.’s laboratory is supported by Medical Research Council (G00900901), Biotechnology and Biological Sciences Research Council (BB/1021833) and The Wellcome Trust (092071/Z/10/Z). E.G. held an MRC Doctoral Studentship; S.H. is supported from Daiichi Sankyo Foundation of Life Science, Japan, L.U. is supported by OXION Wellcome Trust Initiative, Oxford. Y.K. is supported from the Pennsylvania Department of Health using Tobacco CURE Funds SAP#4100062216; P.K. from National Institutes of Health (NIH) R01DC009607 and a visiting Fellowship at St. Catherine’s College, Oxford. F.C.’s laboratory is supported by Human Brain Project (European Flagship, Ref. GA 604102 and Ministerio de Economia y Competitividad MINECO (Spain; Grant BFU2017-88549-P)

A Designer AAV Variant Permits Efficient Retrograde Access to Projection Neurons

Efficient retrograde access to projection neurons for the delivery of sensors and effectors constitutes an important and enabling capability for neural circuit dissection. Such an approach would also be useful for gene therapy, including the treatment of neurodegenerative disorders characterized by pathological spread through functionally connected and highly distributed networks. Viral vectors, in particular, are powerful gene delivery vehicles for the nervous system, but all available tools suffer from inefficient retrograde transport or limited clinical potential. To address this need, we applied in vivo directed evolution to engineer potent retrograde functionality into the capsid of adeno-associated virus (AAV), a vector that has shown promise in neuroscience research and the clinic. A newly evolved variant, rAAV2-retro, permits robust retrograde access to projection neurons with efficiency comparable to classical synthetic retrograde tracers and enables sufficient sensor/effector expression for functional circuit interrogation and in vivo genome editing in targeted neuronal populations. VIDEO ABSTRACT

High-performance probes for light and electron microscopy

We describe an engineered family of highly antigenic molecules based on GFP-like fluorescent proteins. These molecules contain numerous copies of peptide epitopes and simultaneously bind IgG antibodies at each location. These “spaghetti monster” fluorescent proteins (smFPs) distribute well in neurons, notably into small dendrites, spines and axons. smFP immunolabeling localizes weakly expressed proteins not well resolved with traditional epitope tags. By varying epitope and scaffold, we generated a diverse family of mutually orthogonal antigens. In cultured neurons and mouse and fly brains, smFP probes allow robust, orthogonal multi-color visualization of proteins, cell populations and neuropil. smFP variants complement existing tracers, greatly increase the number of simultaneous imaging channels, and perform well in advanced preparations such as array tomography, super-resolution fluorescence imaging and electron microscopy. In living cells, the probes improve single-molecule image tracking and increase yield for RNA-Seq. These probes facilitate new experiments in connectomics, transcriptomics and protein localization

Shared and distinct transcriptomic cell types across neocortical areas

The neocortex contains a multitude of cell types that are segregated into layers and functionally distinct areas. To investigate the diversity of cell types across the mouse neocortex, here we analysed 23,822 cells from two areas at distant poles of the mouse neocortex: the primary visual cortex and the anterior lateral motor cortex. We define 133 transcriptomic cell types by deep, single-cell RNA sequencing. Nearly all types of GABA (γ-aminobutyric acid)-containing neurons are shared across both areas, whereas most types of glutamatergic neurons were found in one of the two areas. By combining single-cell RNA sequencing and retrograde labelling, we match transcriptomic types of glutamatergic neurons to their long-range projection specificity. Our study establishes a combined transcriptomic and projectional taxonomy of cortical cell types from functionally distinct areas of the adult mouse cortex.We thank M. Chillon Rodrigues for providing CAV2-Cre, A. Karpova for providing rAAV2-retro, A. Williford for technical assistance, and the Transgenic Colony Management and Animal Care teams for animal husbandry. This work was funded by the Allen Institute for Brain Science, and by US National Institutes of Health grants R01EY023173 and U01MH105982 to H.Z. We thank the Allen Institute founder, P. G. Allen, for his vision, encouragement and support. (Allen Institute for Brain Science; R01EY023173 - US National Institutes of Health; U01MH105982 - US National Institutes of Health)Accepted manuscrip

Genomic analysis of early SARS-CoV-2 breakthrough infections from the state of Kerala suggest a preponderance of variants of concern

The SARS-CoV-2 Variant of Concern, Delta (B.1.617.2) was first reported in December 2020 in India and has spread colossally throughout the globe. Owing to factors like increased transmissibility, immune escape, and virulence, the delta variant has been considered as a potential public health threat apart from other variants of concern like alpha, beta and gamma. Kerala was one of the first states in India to enroll in the systematic genomic surveillance. In the present report, vaccine breakthrough infections were followed up in 147 patients including 55 healthcare workers who had been vaccinated with ChAdOx1 nCoV- 19/BBV152 across eleven districts from the state of Kerala. The timeline of samples analysed were from April 2021 till June 2021. Severity of the infections reported in the enrolled patients found to be mildly symptomatic, majorly with only 0.7% (n=1) of the cohort to be asymptomatic. Genomic analysis of the samples revealed the Delta variant (B.1.617.2) to constitute about 81.6% (n=120) in the studied cohort. This was followed by the Kappa variant B.1.617.1 (8.35%, n=9), AY.1 (0.6%, n= 1), AY.12 (0.6%, n= 1), AY.4 (1.2%, n= 2), AY.9 (1.2%, n= 2) and Eta variant, B.1.525 (0.6%, n= 1). 11 samples were not assigned any lineage. Evidence from this study suggests the preponderance of the Delta variant in the samples analysed