Spinal cord injury leads to severe problems involving impaired motor, sensory, and autonomic functions. After spinal injury there is an initial phase of hyporeflexia followed by hyperreflexia, often referred to as spasticity. Previous studies have suggested a relationship between the reappearance of endogenous plateau potentials in motor neurons and the development of spasticity after spinalization. To unravel the molecular mechanisms underlying the increased excitability of motor neurons and the return of plateau potentials below a spinal cord injury we investigated changes in gene expression in this cell population. We adopted a rat tail-spasticity model with a caudal spinal transection that causes a progressive development of spasticity from its onset after 2 to 3 wk until 2 mo postinjury. Gene expression changes of fluorescently identified tail motor neurons were studied 21 and 60 days postinjury. The motor neurons undergo substantial transcriptional regulation in response to injury. The patterns of differential expression show similarities at both time points, although there are 20% more differentially expressed genes 60 days compared with 21 days postinjury. The study identifies targets of regulation relating to both ion channels and receptors implicated in the endogenous expression of plateaux. The regulation of excitatory and inhibitory signal transduction indicates a shift in the balance toward increased excitability, where the glutamatergic N-methyl-d-aspartate receptor complex together with cholinergic system is up-regulated and the gamma-aminobutyric acid type A receptor system is down-regulated. The genes of the pore-forming proteins Cav1.3 and Nav1.6 were not up-regulated, whereas genes of proteins such as nonpore-forming subunits and intracellular pathways known to modulate receptor and channel trafficking, kinetics, and conductivity showed marked regulation. On the basis of the identified changes in global gene expression in motor neurons, the present investigation opens up for new potential targets for treatment of motor dysfunction following spinal cord injury.

BackgroundSpinal cord injury leads to neurological dysfunctions affecting the motor, sensory as well as the autonomic systems. Increased excitability of motor neurons has been implicated in injury-induced spasticity, where the reappearance of self-sustained plateau potentials in the absence of modulatory inputs from the brain correlates with the development of spasticity.ResultsHere we examine the dynamic transcriptional response of motor neurons to spinal cord injury as it evolves over time to unravel common gene expression patterns and their underlying regulatory mechanisms. For this we use a rat-tail-model with complete spinal cord transection causing injury-induced spasticity, where gene expression profiles are obtained from labeled motor neurons extracted with laser microdissection 0, 2, 7, 21 and 60 days post injury. Consensus clustering identifies 12 gene clusters with distinct time expression profiles. Analysis of these gene clusters identifies early immunological/inflammatory and late developmental responses as well as a regulation of genes relating to neuron excitability that support the development of motor neuron hyper-excitability and the reappearance of plateau potentials in the late phase of the injury response. Transcription factor motif analysis identifies differentially expressed transcription factors involved in the regulation of each gene cluster, shaping the expression of the identified biological processes and their associated genes underlying the changes in motor neuron excitability.ConclusionsThis analysis provides important clues to the underlying mechanisms of transcriptional regulation responsible for the increased excitability observed in motor neurons in the late chronic phase of spinal cord injury suggesting alternative targets for treatment of spinal cord injury. Several transcription factors were identified as potential regulators of gene clusters containing elements related to motor neuron hyper-excitability, the manipulation of which potentially could be used to alter the transcriptional response to prevent the motor neurons from entering a state of hyper-excitability.

BackgroundIn the field of neuroscience microarray gene expression profiles on anatomically defined brain structures are being used increasingly to study both normal brain functions as well as pathological states. Fluorescent tracing techniques in brain tissue that identifies distinct neuronal populations can in combination with global gene expression profiling potentially increase the resolution and specificity of such studies to shed new light on neuronal functions at the cellular level.Methodology/Principal FindingsWe examine the microarray gene expression profiles of two distinct neuronal populations in the spinal cord of the neonatal rat, the principal motor neurons and specific interneurons involved in motor control. The gene expression profiles of the respective cell populations were obtained from amplified mRNA originating from 50–250 fluorescently identified and laser microdissected cells. In the data analysis we combine a new microarray normalization procedure with a conglomerate measure of significant differential gene expression. Using our methodology we find 32 genes to be more expressed in the interneurons compared to the motor neurons that all except one have not previously been associated with this neuronal population. As a validation of our method we find 17 genes to be more expressed in the motor neurons than in the interneurons and of these only one had not previously been described in this population.Conclusions/SignificanceWe provide an optimized experimental protocol that allows isolation of gene transcripts from fluorescent retrogradely labeled cell populations in fresh tissue, which can be used to generate amplified aRNA for microarray hybridization from as few as 50 laser microdissected cells. Using this optimized experimental protocol in combination with our microarray analysis methodology we find 49 differentially expressed genes between the motor neurons and the interneurons that reflect the functional differences between these two cell populations in generating and transmitting the motor output in the rodent spinal cord.

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