Ryge Jesper, Westerdahl Ann-Charlotte, Alstrøm Preben, Kiehn Ole
Mammalian Locomotor Laboratory, Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden.
PLoS One. 2008;3(10):e3415. doi: 10.1371/journal.pone.0003415. Epub 2008 Oct 15.
In 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 FINDINGS: We 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/SIGNIFICANCE: We 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.
在神经科学领域，利用解剖学定义的脑结构上的微阵列基因表达谱来研究正常脑功能以及病理状态的情况越来越多。脑组织中的荧光追踪技术可识别不同的神经元群体，与全基因组表达谱分析相结合，可能会提高此类研究的分辨率和特异性，从而在细胞水平上为神经元功能提供新的见解。
方法/主要发现：我们研究了新生大鼠脊髓中两个不同神经元群体的微阵列基因表达谱，即主要运动神经元和参与运动控制的特定中间神经元。从50 - 250个经荧光鉴定和激光显微切割的细胞来源的扩增mRNA中获得各细胞群体的基因表达谱。在数据分析中，我们将一种新的微阵列标准化程序与显著差异基因表达的综合测量方法相结合。使用我们的方法，我们发现与运动神经元相比，中间神经元中有32个基因表达更高，其中除一个基因外，之前均未与该神经元群体相关联。作为对我们方法的验证，我们发现有17个基因在运动神经元中的表达高于中间神经元，其中只有一个基因之前未在该群体中被描述过。
结论/意义：我们提供了一种优化的实验方案，可从新鲜组织中荧光逆行标记的细胞群体中分离基因转录本，该方案可用于从少至50个激光显微切割细胞中生成用于微阵列杂交的扩增aRNA。将这种优化的实验方案与我们的微阵列分析方法相结合，我们发现运动神经元和中间神经元之间有49个差异表达基因，这些基因反映了这两种细胞群体在啮齿动物脊髓中产生和传递运动输出方面的功能差异。
