The Role Of Glycine Receptor α2 Subtype In The Proliferation Of The Adult Neuroprogenitor Cells

Since the 1960s and 1970s,Altman reported for the first time that the adult mammalian brain existed neurogenesis,a lot of studies found that there were some neuroprogenitor cells which have the potential of differentiating towards neurons and glial cells in the adult birds,rodents,marmoset and human brain,it could generate new neurons to adapt to certain physical reaction(such as learning,movement,stress, baby identification) or pathological reaction(such as cerebral ischemia,depression, schizophrenia,epilepsy,etc.).This phenomenon is known as adult neurogenesis. Neurogenesis occurs in the adult brain in at least two regions,the SVZ and the SGZ. It has been estimated that 30,000 cells are generated bilaterally daily in the mouse SVZ,which migrated following the rostral migration stream(RMS),then differentiated into interneurons;while 9,000 cells are generated bilaterally daily in the mouse SGZ,which migrated towards granule cell layer to differentiate to granule cells.Until recently the studies found that some areas in the brain,such as striatum, cortex,pyramidal cell layer in hippocampal CA1 region didn’t have neurogenesis under physiological conditions,but under pathological conditions(such as stroke), these areas could generate neurogenesis.Adult neurogenensis included many components:proliferation,migration,differentiation,maturation,survival of adult neuroprogenitor cells and establishment of functional integration.Factors could regulate adult neurogenesis by acting on these different components.In recent years the studies showed that adult neurogenesis would bring new hope to the treatment of brain damage diseases and mental illnesses.But the biological characteristics of adult neuroprogenitor cells were unknown,and the cellular and molecular mechanisms of adult neurogenesis were unknown too?The glycine receptor(GlyR) is a member of pentameric ligand-gated chloride channel,which is homopolymer composed ofαsubunits or heteropolymer composed of twoαsubunits and three 13 subunits.As we know,αsubunits have four subtypes:α1,α2,α3,α4;βsubunit has one subtype.While theα-subunit genes are highly homologous,with primary structures displaying 80-90%amino acid sequence identity,theβ-subunit has a sequence similarity of 47%with theα-subunit. Interestingly,the composition and distribution of glycine receptor has transformed during development.The neonatal form of the GlyR is thought to be a homopentamer ofα2 subunits that mainly found extrasynaptically in vivo,whereas adult synaptic GlyRs are composed ofα1 andβsubunits.Prenatally,transcripts of theα2-subunit gene are found throughout most of the central nervous system;postnatally they decline sharply with little label remaining,however,significant expression was still seen in many adult brain regions including hippocampus,thalamus et al.These studies showed that GlyRα2 subtype might play role in the development of cells.In the last few years,a great deal of studies showed that glycine receptorα2 subunit had important functions in regulating the development of neurons.In 1998, Flint group reported that taurine is released non-synaptically from immature cortical neurons,which has been found to influence cortical development by activating extrasynaptic GlyRs.And taurine deprivation of kitten in utero leads to low brain weight and an apparent disruption of normal cortical migration and differentiation.In 2004,Young and Cepko reported that targeted knockdown of GlyRa2 subunit decreased the number of photoreceptors in the developing retina,the GlyRa2 subunit was expressed by retinal progenitor cells.In 2006,Pierre Drapeau group reported that targeted knockdown of the embryonic GlyRa2 subunit disrupted rhythm-generating networks,immunohistochemistry revealed a reduction in the number of spinal interneurons.These studies implicate extrasynaptic GlyRs can regulate the development of neurons.The results of our previous studies also showed that:adult neuroprogenitor cells expressed GlyRa2 subunit;the antagonist of glycine receptor could decrease the numbers of newborn neurons in SGZ,while agonist of glycine receptor could increase the numbers of newborn neurons,these results implicate that glycine receptor may participate in the regulation of adult neuroprogenitor cells.Now the purpose of our study is to prove this phenomenon in vitro and study the mechanisms.To study the factors which influence the proliferation of adult neuroprogenitor cells,we need to culture the adult neuroprogenitor cells in vitro,but the culture of adult neuroprogenitor cells is not always easy.Now,there were two methods about the culture of adult neuroprogenitor cells in the world:suspend culture and attachment culture.The method of attachment was only possessed by Gage’s group, and was accepted by most students in the world.The main purpose of our study is to culture adult neuroprogenitor cells in vitro, and to identify the glycine receptor subunits in adult neuroprogenitor cells,in particularα2 subunit,and to observe the effect of glycine receptor antagonists Strychnine、Picrotoxin and agonist Taurine on the proliferation of adult neuroprogenitor cells,then to explore the role of glycine receptorα2 subtype in regulating the proliferation of adult neuroprogenitor cells.The adult neuroprogenitor cells were cultured in serum-free rat neural stem cell expansion medium containing basic fibroblast growth factor(bFGF).Our study found that adult neuroprogenitor cells were small、round with thin processes.The proportion of nestin-positive cells reached to 99%The expression of Glycine receptor subunits:the results of immunofluorescence and RT-PCR showed that glycine receptorα2 subunit expressed in adult neuroprogenitor cells,but none expression ofα1 andα3 subunit.To study the regulation of glycine receptor in adult neuroprogenitor cells: the single-cell suspensions were cultured on the Polylysine-coated culture plank, then to add different concentrations of Strychnine,Picrotoxin,Taurine to culture medium for 46 hours,then 5 umol/L BrdU for two hours,finally immunofluorescence labeled the percent of BrdU/DAPI co-positive cells.The percent of BrdU-positive cells decrease to 88.75%,86.66%、81.63%、80.39%compare 2.5uM Strychnine group、5 uM Strychnine group,10 uM Strychnine group,20 uM Strychnine group with the control group,there had statistically significances.And the percent of BrdU-positive cells decrease to 104.32%,88.2%,73.77%,66.84%compare 5uM Picrotoxin group,10uM Picrotoxin group、20uM Picrotoxin group,40uM Picrotoxin group with the control group,there had statistically significances.Compared the 200uM Taurine group、600 uM Taurine,1mM Taurine group with the control group, there had no statistically significances.The results showed that Strychnine and Picrotoxin could inhibit the proliferation of adult neuroprogenitor cells by a concentration-dependent manner.The experimental results showed that glycine receptor ct2 subunit was expressed on adult neuroprogenitor cells,and the glycine receptor antagonist Strychnine, Picrotoxin could inhibit the proliferation of adult neuroprogenitor cells,which suggested that glycine receptorα2 subtype might be involved in the regulation of proliferation of adult neuroprogenitor cells.