Activation of MAP2K signaling by genetic engineering or HF-rTMS promotes corticospinal axon sprouting and functional regeneration.

Facilitating axon regeneration in the injured central nervous system remains a challenging task. RAF-MAP2K signaling plays a key role in axon elongation during nervous system development. Here, we show that conditional expression of a constitutively kinase-activated BRAF in mature corticospinal neurons elicited the expression of a set of transcription factors previously implicated in the regeneration of zebrafish retinal ganglion cell axons and promoted regeneration and sprouting of corticospinal tract (CST) axons after spinal cord injury in mice. Newly sprouting axon collaterals formed synaptic connections with spinal interneurons, resulting in improved recovery of motor function. Noninvasive suprathreshold high-frequency repetitive transcranial magnetic stimulation (HF-rTMS) activated the BRAF canonical downstream effectors MAP2K1/2 and modulated the expression of a set of regeneration-related transcription factors in a pattern consistent with that induced by BRAF activation. HF-rTMS enabled CST axon regeneration and sprouting, which was abolished in MAP2K1/2 conditional null mice. These data collectively demonstrate a central role of MAP2K signaling in augmenting the growth capacity of mature corticospinal neurons and suggest that HF-rTMS might have potential for treating spinal cord injury by modulating MAP2K signaling.

A community-based transcriptomics classification and nomenclature of neocortical cell types.

To understand the function of cortical circuits, it is necessary to catalog their cellular diversity. Past attempts to do so using anatomical, physiological or molecular features of cortical cells have not resulted in a unified taxonomy of neuronal or glial cell types, partly due to limited data. Single-cell transcriptomics is enabling, for the first time, systematic high-throughput measurements of cortical cells and generation of datasets that hold the promise of being complete, accurate and permanent. Statistical analyses of these data reveal clusters that often correspond to cell types previously defined by morphological or physiological criteria and that appear conserved across cortical areas and species. To capitalize on these new methods, we propose the adoption of a transcriptome-based taxonomy of cell types for mammalian neocortex. This classification should be hierarchical and use a standardized nomenclature. It should be based on a probabilistic definition of a cell type and incorporate data from different approaches, developmental stages and species. A community-based classification and data aggregation model, such as a knowledge graph, could provide a common foundation for the study of cortical circuits. This community-based classification, nomenclature and data aggregation could serve as an example for cell type atlases in other parts of the body.

Semantic segmentation of microscopic neuroanatomical data by combining topological priors with encoder-decoder deep networks.

Understanding of neuronal circuitry at cellular resolution within the brain has relied on neuron tracing methods which involve careful observation and interpretation by experienced neuroscientists. With recent developments in imaging and digitization, this approach is no longer feasible with the large scale (terabyte to petabyte range) images. Machine learning based techniques, using deep networks, provide an efficient alternative to the problem. However, these methods rely on very large volumes of annotated images for training and have error rates that are too high for scientific data analysis, and thus requires a significant volume of human-in-the-loop proofreading. Here we introduce a hybrid architecture combining prior structure in the form of topological data analysis methods, based on discrete Morse theory, with the best-in-class deep-net architectures for the neuronal connectivity analysis. We show significant performance gains using our hybrid architecture on detection of topological structure (e.g. connectivity of neuronal processes and local intensity maxima on axons corresponding to synaptic swellings) with precision/recall close to 90% compared with human observers. We have adapted our architecture to a high performance pipeline capable of semantic segmentation of light microscopic whole-brain image data into a hierarchy of neuronal compartments. We expect that the hybrid architecture incorporating discrete Morse techniques into deep nets will generalize to other data domains.

SEMA3A signaling controls layer-specific interneuron branching in the cerebellum.

BACKGROUND: GABAergic interneurons regulate the balance and dynamics of neural circuits, in part, by elaborating their strategically placed axon branches that innervate specific cellular and subcellular targets. However, the molecular mechanisms that regulate target-directed GABAergic axon branching are not well understood. RESULTS: Here we show that the secreted axon guidance molecule, SEMA3A, expressed locally by Purkinje cells, regulates cerebellar basket cell axon branching through its cognate receptor Neuropilin-1 (NRP1). SEMA3A was specifically localized and enriched in the Purkinje cell layer (PCL). In sema3A(-/-) and nrp1(sema-/sema-) mice lacking SEMA3A-binding domains, basket axon branching in PCL was reduced. We demonstrate that SEMA3A-induced axon branching was dependent on local recruitment of soluble guanylyl cyclase (sGC) to the plasma membrane of basket cells, and sGC subcellular trafficking was regulated by the Src kinase FYN. In fyn-deficient mice, basket axon terminal branching was reduced in PCL, but not in the molecular layer. CONCLUSIONS: These results demonstrate a critical role of local SEMA3A signaling in layer-specific axonal branching, which contributes to target innervation.

New insights into the classification and nomenclature of cortical GABAergic interneurons.

A systematic classification and accepted nomenclature of neuron types is much needed but is currently lacking. This article describes a possible taxonomical solution for classifying GABAergic interneurons of the cerebral cortex based on a novel, web-based interactive system that allows experts to classify neurons with pre-determined criteria. Using Bayesian analysis and clustering algorithms on the resulting data, we investigated the suitability of several anatomical terms and neuron names for cortical GABAergic interneurons. Moreover, we show that supervised classification models could automatically categorize interneurons in agreement with experts' assignments. These results demonstrate a practical and objective approach to the naming, characterization and classification of neurons based on community consensus.

Correlation between axonal morphologies and synaptic input kinetics of interneurons from mouse visual cortex.

Neocortical interneurons display great morphological and physiological variability and are ideally positioned to control circuit dynamics, although their exact role is still poorly understood. To better understand this diversity, we have performed a detailed anatomical and physiological characterization of 3 subtypes of visual cortex interneurons, isolated from transgenic mice which express green fluorescent protein in somatostatin, parvalbumin, and neuropeptide Y positive neurons. We find that these 3 groups of interneurons have systematic differences in dendritic and axonal morphologies and also characteristically differ in the frequencies, amplitude, and kinetics of the spontaneous excitatory and inhibitory synaptic currents they receive. Moreover, we detect a correlation between the kinetics of their synaptic inputs and quantitative aspects of their axonal arborizations. This suggests that different interneuron types could channel different temporal patterns of activity. Our results also confirm the importance of the axonal morphology to classify interneurons.

Cytosolic branched chain aminotransferase (BCATc) mRNA is up-regulated in restricted brain areas of BDNF transgenic mice.

Branched chain aminotransferase (BCAT) catalyzes the transamination of the essential branched chain amino acids (leucine, isoleucine and valine) with alpha-ketoglutarate. BCAT exists in two isoforms: one cytosolic (BCATc), mainly expressed in the nervous system, and the other mitochondrial (BCATm), present in a greater number of tissues. We previously showed that BCATc mRNA and protein expression in the dorsal lateral geniculate nucleus of the thalamus is up-regulated by exogenous administration of brain-derived neurotrophic factor (BDNF) following lesion of the visual cortex in newborn rats. Here, we analyzed the expression of BCATc mRNA in the brain of transgenic mice overexpressing the rat BDNF cDNA under the control of the alpha-calcium/calmodulin-dependent kinase II (alphaCaMKII) promoter. In these animals, BDNF is overexpressed in the telencephalon starting from the second postnatal week. RT-PCR and in situ hybridization experiments showed that BCATc mRNA is overexpressed in restricted regions of the cerebral cortex (parietal area) and hippocampus (hilus and CA3 pyramidal cell layer) of adult BDNF transgenic mice respect to wild-type animals. These differences between wt and BDNF mice were not detected in animals of 1 week of age. These results demonstrate that the expression of the BCATc gene in the brain is specifically regulated by BDNF in a time- and region-dependent fashion.

Neural cell adhesion molecule-secreting transgenic mice display abnormalities in GABAergic interneurons and alterations in behavior.

The extracellular region of the transmembrane neural cell adhesion molecule (NCAM-EC) is shed as a soluble fragment at elevated levels in the schizophrenic brain. A novel transgenic mouse line was generated to identify consequences on cortical development and function of expressing soluble NCAM-EC from the neuron-specific enolase promoter in the developing and mature neocortex and hippocampus. NCAM-EC transgenic mice exhibited a striking reduction in synaptic puncta of GABAergic interneurons in the cingulate, frontal association cortex, and amygdala but not hippocampus, as shown by decreased immunolabeling of glutamic acid decarboxylase-65 (GAD65), GAD67, and GABA transporter 1. Interneuron cell density was unaltered in the transgenic mice. Affected subpopulations of interneurons included basket interneurons evident in NCAM-EC transgenic mice intercrossed with a reporter line expressing green fluorescent protein and by parvalbumin staining. In addition, there appeared to be a reduction in excitatory synapses, as revealed by synaptophysin staining and apical dendritic spine density of cortical pyramidal cells. Behavioral analyses demonstrated higher basal locomotor activity of NCAM-EC mice and enhanced responses to amphetamine and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate compared with wild-type controls. Transgenic mice were deficient in prepulse inhibition, which was restored by clozapine but not by haloperidol. Additionally, NCAM-EC mice were impaired in contextual and cued fear conditioning. These results suggested that elevated shedding of NCAM perturbs synaptic connectivity of GABAergic interneurons and produces abnormal behaviors that may be relevant to schizophrenia and other neuropsychiatric disorders.