ABSTRACT
Injury of CNS nerve tracts remodels circuitry through dendritic spine loss and hyper-excitability, thus influencing recovery. Due to the complexity of the CNS, a mechanistic understanding of injury-induced synaptic remodeling remains unclear. Using microfluidic chambers to separate and injure distal axons, we show that axotomy causes retrograde dendritic spine loss at directly injured pyramidal neurons followed by retrograde presynaptic hyper-excitability. These remodeling events require activity at the site of injury, axon-to-soma signaling, and transcription. Similarly, directly injured corticospinal neurons in vivo also exhibit a specific increase in spiking following axon injury. Axotomy-induced hyper-excitability of cultured neurons coincides with elimination of inhibitory inputs onto injured neurons, including those formed onto dendritic spines. Netrin-1 downregulation occurs following axon injury and exogenous netrin-1 applied after injury normalizes spine density, presynaptic excitability, and inhibitory inputs at injured neurons. Our findings show that intrinsic signaling within damaged neurons regulates synaptic remodeling and involves netrin-1 signaling.Spinal cord injury can induce synaptic reorganization and remodeling in the brain. Here the authors study how severed distal axons signal back to the cell body to induce hyperexcitability, loss of inhibition and enhanced presynaptic release through netrin-1.
Subject(s)
Dendritic Spines/physiology , Netrin-1/metabolism , Neuronal Plasticity , Pyramidal Cells/physiology , Synapses/physiology , Animals , Axotomy , Embryo, Mammalian , Gene Expression , Glutamic Acid/metabolism , Microfluidic Analytical Techniques , Motor Cortex/physiopathology , Primary Cell Culture , Rats, Sprague-Dawley , Spinal Cord Injuries/physiopathologyABSTRACT
The identification of mRNAs in distal projections of model organisms has led to the discovery of multiple proteins that are locally synthesized for functional roles such as axon guidance, injury signaling and regeneration. The extent to which local protein synthesis is conserved in human neurons is unknown. Here we used compartmentalized microfluidic chambers to characterize the transcriptome of distal projections of human embryonic stem cells differentiated using a protocol which enriched for glutamatergic neurons (hESC-neurons). Using gene expression analysis, we identified mRNAs proportionally enriched in these projections, representing a functionally unique local transcriptome as compared to the human neuronal transcriptome inclusive of somata. Further, we found that the most abundant mRNAs within these hESC-neuron projections were functionally similar to the axonal transcriptome of rat cortical neurons. We confirmed the presence of two well characterized axonal mRNAs in model organisms, ß-actin and GAP43, within hESC-neuron projections using multiplexed single molecule RNA-FISH. Additionally, we report the novel finding that oxytocin mRNA localized to these human projections and confirmed its localization using RNA-FISH. This new evaluation of mRNA within human projections provides an important resource for studying local mRNA translation and has the potential to reveal both conserved and unique translation dependent mechanisms.
