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 [4].

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 [5]. Thermoresponsive hydrogel scaffolds have been introduced into primary embryonic cortical neuron cultures to repair spinal cord injuries [6]. 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 [11]. 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 [12]. 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 [16]. 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 [22]. 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 [23], 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 [24]. 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 [28] and has four transmembrane domains including synaptogyrin and synaptoporin [29]. It has been suggested that synaptophysin promotes the formation of highly curved membranes such as synaptic vesicles [30]. An ultra-structural study demonstrated that synaptophysin forms a structure similar to connexons [31]. Synaptophysin was recently reported to regulate the kinetics of synaptic vesicular endocytosis in neurons [32].

In this study, we focused on neurite outgrowth and synaptogenic activity using immunofluorescence and Western blotting with synaptogenic markers during the early period of in vitro culture. We aimed to demonstrate whether the patterns of neurite outgrowth were consistent with the expression levels of neurite outgrowth and synaptogenic markers by comparing morphological findings with those of Western blotting. Data from this study will be a useful reference for many primary neuronal culture protocols, because the primary culture system used in this study is a feasible in vitro model for a wide variety of neurophysiological and neuropharmacological applications.

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 in vitro by using one of the well known neurite outgrowth and synaptogenesis markers and the detailed results are as follows.

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 [33]. Interestingly, the expression of synaptic markers from cortical neurons reached a peak level earlier (around 5 days in vitro) than that from hippocampal neurons (10 days in vitro), although neurite outgrowth from hippocampal neurons was faster than that of cortical neurons. Furthermore, the peak amounts of expressed synaptic markers were also higher in cortical neurons than those in hippocampal neurons. Mice embryos at embryonic days 16~18 were used, although postnatal new-born rodents could be also used for primary neuronal or neural stem cell cultures [34-36]. The matrigel that we used for coating the glass cover slips creates more optimal conditions for survival and neurite outgrowth than coating with laminin or other substrates alone. The majority of plated neurons did not survive on non-coated cover slips and clumped heavily (data not shown). We found a gradual increase in GAP-43 immunoreactivity that was associated with extension and branching of neurites. Synaptophysin immunoreactivity was already strong enough from day 2 in vitro. Although we are unable to confirm whether they are true synaptic buttons, these findings could be considered important because the more synaptic buttons secured, the better the synaptic functional or morphological study. GAP-43 is a marker of synaptic plasticity that is critical for normal development of serotonergic innervation [37], and synaptophysin is a well-known synaptic vesicular protein. Considering prompt neurite outgrowth and abundant branching, synaptic marker expression levels should be clarified, particularly during the early period in vitro. Our Western blotting results seemed to be a useful reference for experimental designs on synaptic activity and plasticity.

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 in vitro) in functional studies associated with synaptogenesis. Although we are unable to suggest a possible explanation for this differential expression, the expression of synaptic vesicular associated proteins from cortical neurons seemed to be accelerated within the first week in vitro as a preparatory stage for explosive synaptogenic processes thereafter. To explain these discrepancies between neuronal groups during the early in vitro period, we are going to introduce primary cultured neurons that lack synaptic markers or synaptic vesicular associated proteins (synaptobrevin and Munc-18) as a next step. Although we are only showing findings based on morphological changes and synaptic markers expression at this time, we are planning to identify more detailed clues or mechanisms of growth cone formation and synaptogenesis during neuronal growth and differentiation by adding neurotrophic factors such brain derived neurotrophic factor (BDNF). Findings from peripheral nerve cell and neuronal stem cell cultures should also be compared to determine whether they have different mechanisms according to their origin.