Gene Expression Profiling of Two Distinct Neuronal Populations in the Rodent Spinal Cord

Abstract

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.

Effects of chronic spinal cord injury on relationships among ion channel and receptor mRNAs in mouse lumbar spinal cord

“…Second, we see widespread changes in relative abundance of a given transcript following SCI. It is clear that SCI causes widespread changes in gene expression in the spinal cord across numerous gene families: this has been demonstrated through the use of qPCR (Esmaeili and Zaker, 2011;Di Narzo et al, 2015), microarrays (Carmel et al, 2001;Ryge et al, 2008Ryge et al, , 2010Wienecke et al, 2010;Liu et al, 2014) and RNAseq (Chen et al, 2013;Lee-Liu et al, 2014). Our results add to this body of work, and can help provide insight into potential mechanisms underlying changes in physiology following SCI.…”

“…It is clear that spinal cord injury causes widespread changes in gene expression of the spinal cord across numerous gene families; this has been demonstrated by various approaches including qPCR (Esmaeili and Zaker, 2011;Di Narzo et al, 2015), microarray analyses (Carmel et al, 2001;Ryge et al, 2008Ryge et al, , 2010Wienecke et al, 2010;Liu et al, 2014) and RNAseq (Chen et al, 2013;Lee-Liu et al, 2014). There is also growing appreciation that in addition to overall levels of transcript, expression levels of some genes vary in parallel over repeated measurements: this co-regulation of functionally interacting channels and receptors may be essential to maintain appropriate neuronal output (Amendola et al, 2012).…”

Weight-Bearing Locomotion in the Developing Opossum, Monodelphis domestica following Spinal Transection: Remodeling of Neuronal Circuits Caudal to Lesion

“…A further contrast suggesting an increase in Gria2 and Grm5 in the L3–5 segments of P28-injured animals suggests that significant reorganization occurs not only between ages but also along the rostrocaudal axis of the cord. Gria2 has been shown to be expressed more highly in interneurons than motoneurons in the lumbar cord [52], pointing to a potential difference in the balance of these neuronal subtypes in the lower lumbar segments of P28-injured animals.…”

“…However there was a lack of consistent and repeatable hybridization with Monodelphis cDNA, leading to large variability in control values so that no statistical differences between operated and control animals could be detected. Other studies using more conventional laboratory species have successfully employed genetic screens (usually in the form of microarrays [40]–[43], [52]). We limited our studies to three representative genes from each category of transmitters: excitatory, inhibitory and neuromodulatory.…”

Transcriptional regulation of gene expression clusters in motor neurons following spinal cord injury

“…A conglomerate classifier based on three well-established adjusted ANOVA test-statistics for microarray analysis (limma, Cyber-T and SAM) was used to identify significantly differentially expressed genes used for subsequent clustering, identifying 3,708 genes with a set false discovery rate (FDR) threshold of 0.02 [34]. …”