ASMS Conference on Mass Spectrometry edition:53 location:San Antonio, Texas, U.S.A. date:June 5-9, 2005
Molecular differences between developmental and adult brain plasticity models in mice using 2D-DIGE and MS
Within a ‘critical period’ early in life, all sensory systems in the brain of mammals have the remarkable capacity to adapt to changes in sensory input by modifying neuronal connectivity. The goal-directed strengthening, remodelling and elimination of synapses create the adult-specific neuronal circuitry. Later in life this plasticity appears greatly reduced. The molecular mechanisms underlying this age-dependent difference in brain plasticity is not yet known in great detail. Using fluorescent 2D differential gel electrophoresis (2D-DIGE) and mass spectrometry, we investigate the (dis)similarities in the molecular mechanisms of brain plasticity during development and adulthood.
As developmental plasticity model, whole forebrain extracts of 10-day-old pups and adult mice were compared. For adult brain plasticity, we focussed on the barrel cortex, a subdivision of the primary somatosensory cortex. Each barrel is related to a single vibrissa on the contralateral face. Adult mice were sensory deprived by removing the vibrissae on the right side of the snout, and killed after 2 weeks in an enriched environment. After brain removal and snap-freezing, cryostat sections were made and barrels were visualised via cytochrome oxidase staining. Proteins were extracted from barrel cortex of deprived and non-deprived hemispheres. Paired samples were analysed by 2D-DIGE. Spots were visualised, differential spots were excised from gel, and identified via MALDI-TOF MS or Q-TOF MS/MS.
Protein samples from 2 different conditions were labelled with two different fluorescent dyes, Cy3 and Cy5 and consecutively paired. Reverse labelling was carried out to clarify possible dye-specific preferential labelling. The first dimension of the 2D-DIGE experiments was performed on immobilised pH gradients, ranging from pH 4-7 and from 3-10 (non linear). Only spots that switched colour in forward and reverse labelling were considered to contain differentially expressed proteins. The spots visually identified as differential were statistically analysed using Phoretics 2D v2004 spot analysis software. Between forebrain extracts of 10-day-old mouse pups and adult mice 77% of the 45 protein spots with differential colouring (pH 4-7), showed a statistically significant difference in fluorescence level (p<0.05). In the wide pH-gradient gels, almost 50% of 45 differentially coloured spots appeared more abundant in the 10-day-old pups as is currently verified statistically. Excision of the spots for protein identification was done from Coomassie Brilliant Blue stained preparative gels and the spots were identified using MALDI-TOF MS or nanoscale HPLC coupled to Q-TOF MS/MS after in-gel tryptic digestion. Apart from metabolic enzymes, identified spots contained proteins involved in the outgrowth of axons and in growth cone collapse (collapsin response mediator proteins). Moreover they are involved in neuronal differentiation during the development of the nervous system. In adult brain, the protein content of punches of left sensory-deprived and right normal hemisphere barrel cortex was compared. Upon 2D-DIGE, only few protein spots appeared differential. Our first results therefore clearly indicate molecular differences between developmental and adult brain plasticity.