ABSTRACT
Despite substantial progress in understanding the biology of axon regeneration in the CNS, our ability to promote regeneration of the clinically important corticospinal tract (CST) after spinal cord injury remains limited. To understand regenerative heterogeneity, we conducted patch-based single-cell RNA sequencing on rare regenerating CST neurons at high depth following PTEN and SOCS3 deletion. Supervised classification with Garnett gave rise to a Regeneration Classifier, which can be broadly applied to predict the regenerative potential of diverse neuronal types across developmental stages or after injury. Network analyses highlighted the importance of antioxidant response and mitochondrial biogenesis. Conditional gene deletion validated a role for NFE2L2 (or NRF2), a master regulator of antioxidant response, in CST regeneration. Our data demonstrate a universal transcriptomic signature underlying the regenerative potential of vastly different neuronal populations and illustrate that deep sequencing of only hundreds of phenotypically identified neurons has the power to advance regenerative biology.
Subject(s)
Axons , Spinal Cord Injuries , Humans , Axons/physiology , Nerve Regeneration/genetics , Antioxidants , Neurons , Spinal Cord Injuries/genetics , Pyramidal Tracts/physiology , Single-Cell AnalysisABSTRACT
Parvalbumin and somatostatin inhibitory interneurons gate information flow in discrete cortical areas that compute sensory and cognitive functions. Despite the considerable differences between areas, individual interneuron subtypes are genetically invariant and are thought to form canonical circuits regardless of which area they are embedded in. Here, we investigate whether this is achieved through selective and systematic variations in their afferent connectivity during development. To this end, we examined the development of their inputs within distinct cortical areas. We find that interneuron afferents show little evidence of being globally stereotyped. Rather, each subtype displays characteristic regional connectivity and distinct developmental dynamics by which this connectivity is achieved. Moreover, afferents dynamically regulated during development are disrupted by early sensory deprivation and in a model of fragile X syndrome. These data provide a comprehensive map of interneuron afferents across cortical areas and reveal the logic by which these circuits are established during development.
Subject(s)
Cerebral Cortex/pathology , Fragile X Mental Retardation Protein/physiology , Fragile X Syndrome/pathology , Interneurons/pathology , Presynaptic Terminals/pathology , Sense Organs/pathology , Synapses/pathology , Animals , Cerebral Cortex/metabolism , Female , Fragile X Syndrome/genetics , Fragile X Syndrome/metabolism , Interneurons/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neural Pathways , Presynaptic Terminals/metabolism , Rabies virus/genetics , Sense Organs/metabolism , Synapses/metabolismABSTRACT
Optical methods of interrogating neural circuits have emerged as powerful tools for understanding how the brain drives behaviors. Optogenetic proteins are widely used to control neuronal activity, while genetically encoded fluorescent reporters are used to monitor activity. These proteins are often expressed by injecting viruses, which frequently leads to inconsistent experiments due to misalignment of expression and optical components. Here, we describe how silk fibroin films simplify optogenetic experiments by providing targeted delivery of viruses. Films composed of silk fibroin and virus are applied to the surface of implantable optical components. After surgery, silk releases the virus to transduce nearby cells and provide localized expression around optical fibers and endoscopes. Silk films can also be used to express genetically encoded sensors in large cortical regions by using cranial windows coated with a silk/virus mixture. The ease of use and improved performance provided by silk make this a promising approach for optogenetic studies.
