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Exp Neurobiol 2012; 21(2): 61-67
Published online June 30, 2012
© The Korean Society for Brain and Neural Sciences
Yun-Gi Kim1 and Young-Il Lee2*
Departments of 1NanoBio Medical Science, 2Anatomy, College of Medicine, Dankook University, Cheonan 330-714, Korea
Correspondence to: *To whom correspondence should be addressed.
TEL: 82-41-550-3855, FAX: 82-41-556-6461
Primary dissociated neuronal cultures are widely used research tools to investigate of pathological mechanisms and to treat various central and peripheral nervous system problems including trauma and degenerative neuronal diseases. We introduced a protocol that utilizes hippocampal and cortical neurons from embryonic day 17 or 18 mice. We applied appropriate markers (GAP-43 and synaptophysin) to investigate whether neurite outgrowth and synaptogenesis can be distinguished at a particular period of time. GAP-43 was found along the neural processes in a typical granular pattern, and its expression increased proportionally as neurites lengthened during the early
Keywords: primary neuronal culture, mouse embryo, neurite outgrowth, synaptogenesis, gap-43, synaptophysin
Primary neural cultures allow continuous visual access for morphological studies such as neurite sprouting, connectivity, and variability. These cultures make individual living cells accessible to apply chemical or pharmacological agents and patch clamp recording [1-3], which are difficult tasks to perform in a sectioned slice alone. Individual neuronal activity of multiple neurons can be recorded for several weeks, which is extremely difficult to perform in a sectioned slice. Additionally, the relative proportions of neurons and neuroglial cells can be controlled, and different patterns of neuronal connectivity are beginning to be studied with developments in culture substrates. Thus, primary neuronal culture is an important research tool that can be applied on a cell-by-cell basis to morphological and physiological studies of a variety of brain areas, including the cerebral cortex, hippocampal formation, basal ganglia, and the diencephalon. In primary neuronal cultures, newly formed neurons undergo a series of extensive morphological changes as they mature, including neurite sprouting and outgrowth, neurite branching, and establishment of synaptogenesis. Although insufficient, these morphological changes are necessary for the formation of neural network circuits facilitating neuronal functions .
Several researchers have adopted various substrates and methods for primary neuronal or neural stem cell cultures to stimulate and enhance neurite outgrowth. Positively charged hydrogels were used as substrates for nerve cell attachment and neurite outgrowth in rat primary dorsal root ganglion (DRG) cultures . Thermoresponsive hydrogel scaffolds have been introduced into primary embryonic cortical neuron cultures to repair spinal cord injuries . Artificial scaffolds have been investigated as a means of addressing axonal regeneration inhibiting stimuli [7, 8] such as glial scars [9, 10] or inhibiting molecules . Most recently, a three-dimensional microfluidic device showed dissociated cortical neurons cultured in three-dimensional multilayered scaffolds based on an agarose-alginate mixture for the first time . Primary cultures offer an opportunity to visualize neurite outgrowth or synaptogenesis at the level of single cells, and modern immunofluorescent methods are frequently used for this purpose. Because it is inevitable that cells are grown on glass cover slips to prevent quenching or autofluorescence, cover slips must be coated for successful cell attachment and growth. Various compounds, such as poly-l-lysine (PLL), laminin, or poly-l-ornithine have been used for coating [13-15]. Here, we introduce culturing primary cortical and hippocampal neural cells isolated from embryonic mice on cover slips coated with a soluble basement membrane (Matrigel), which contains laminin, collagen IV, heparin sulfate, proteoglycans, entactin, and nidogen . Because dissociated hippocampal or cortical cell cultures are composed of neurons and glial cells, strongly proliferating astrocytes will displace the non-proliferating neurons. Thus, we have added cytostatic drugs, such as cytosine arabinoside (AraC) to growing cells [17-19].
A variety of markers are used widely to detect core matter and core factor during neurite sprouting and outgrowth as well as synaptogenesis. Many researchers who study synapse formation have selected better substances such as axonal membrane protein (GAP-43), synaptophysin, and synapsin [20, 21]. GAP-43 is a phosphoprotein of the nerve terminal membrane that has been linked to the development and restructuring of axons . Recent studies have shown that GAP-43 is not only related to an increase in synapse formation and synaptic plasticity in mice or rat primary neuronal cultures , but it is as also a significant substance for neurite outgrowth as shown by a morphological study with primary neuronal cultures from human fetal forebrain . Moreover, GAP-43 reduction is associated with stress and aging in brain [25-27]. Synaptophysin is one of the synaptic vesicular proteins that has recently been characterized for the first time  and has four transmembrane domains including synaptogyrin and synaptoporin . It has been suggested that synaptophysin promotes the formation of highly curved membranes such as synaptic vesicles . An ultra-structural study demonstrated that synaptophysin forms a structure similar to connexons . Synaptophysin was recently reported to regulate the kinetics of synaptic vesicular endocytosis in neurons .
In this study, we focused on neurite outgrowth and synaptogenic activity using immunofluorescence and Western blotting with synaptogenic markers during the early period of
We used C57/BL6 mouse strain for primary neural culture. Embryonic day 16 to 18 embryos were obtained from surgically sacrificed pregnant mouse and separated cerebral cortex and hippocampus under surgical stereomicroscope. Separated tissues were trypsinized (5 mg/ml) for 10 min in 37℃. Finally, dissociated neurons were cultured on Matrigel (BD Science) coated 12 mm coverslips (total 12 coverslips per cortex or hippocampus). Number of total plated cell was adjusted at around 100,000 per each coverslip. Culture media was prepared based on MEM (minimum essential media) We added glucose (5 gm/l), transferrin (0.1 gm/l), insulin (0.25 gm/l), glutamine (0.3 gm/l), heat-inactivated FBS (5~10%) and B-27 supplement (2%) to MEM as supplements. Culture media was changed only two times in day 1 and day 4 through all
Cover slips those scheduled to be used for immune-fluorescence staining were transferred into 4-well plates in day 2, day 4, and day 8
Confocal microscope (LSM-700 Carl Zeiss, Germany) equipped with associated software of ZEN2009 (version 5,5,0,375 Carl Zeiss, Germany) was used for the analysis of GAP-43 and synaptophysin immunofluorescence staining. We set up the standard master gain of DAPI fixation of 700 (GAP-43 & synaptophysin), master gain of rhodamin fixation of 619 (GAP-43) and 700 (synaptophysin), digital gain below 1 and pinhole below 5.
Cultured neurons were extracted from coverlslips in each wells and homogenized with 5X sample buffer (250 mM Tris-HCl pH 6.8, 30% glycerol, 5% beta-mecaptoethanol, 0.02% bromophenol blue, 10% SDS). After SDS-PAGE, transferred membranes were blocked by 5% skim milk for 30 minutes at RT. For primary antibody reactions, Anti-GAP43 (1:2,000, ab7462, rabbit polyclonal, Abcam) and anti-synaptophysin (1:1,000, ab14692, rabbit polyclonal, Abcam) were added to membranes and stayed overnight at 4℃ on orbital shaker. Anti-beta actin (1:3,000, ab8227, rabbit polyclonal, Abcam) was also added to the membranes as loading controls for 1 hour at RT. After the reaction with goat anti-rabbit (1:3,000, 111-036-003, Jackson) for 1 hour at RT, signals were enhanced by ECL (MC154418, Thermo) solutions. Bands were detected by the image detection system (LAS 4000, GE).
Primary cultured cortical and hippocampal neurons were traced their neurite outgrowth under the phase contrast microscope. Neurons were also tested synaptogenic activities along the time courses of early periods
We investigated morphological changes of primary cultured neurons day by day from day 1
Neurite sprouting and outgrowth of cortical neurons were slower than hippocampal neurons. In many cases, we could find only limited degree of neurite sprouting from cortical neurons in early days
GAP-43 immunoreactivities were gradually increased according to these morphological changes from day 2 to day 8
Along with the immunofluorescence, quantification of synaptic marker expressions was examined by western blotting through the wide range of
We characterized neurite outgrowth and synaptogenic activities in primary cultured cortical and hippocampal neurons using immunofluorescence and Western blotting. The main features of survival, proliferation, and neurite outgrowth were similar to those cultured in a mixture of MEM and B27-supplemented neurobasal medium from rat embryos . Interestingly, the expression of synaptic markers from cortical neurons reached a peak level earlier (around 5 days
We confirmed delayed neurite sprouting and outgrowth of primary cultured cortical neurons compared to those of hippocampal neurons. However, the expression levels of neurite outgrowth and synaptogenesis markers showed reversed patterns between cortical and hippocampal neurons. These results suggest that primary cultured cortical neurons could be utilized rather earlier (within 1 week