Differential Expressions of Synaptogenic Markers between Primary Cultured Cortical and Hippocampal Neurons

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 in vitro period. Unlike GAP-43, granular immunoreactive patterns of synaptophysin along the neurites were clearly found from day 2 in vitro with relatively high immunoreactive levels. Expression of synaptic markers from cortical neurons reached peak level earlier than that of hippocampal neurons, although neurite outgrowths of hippocampal neurons were faster than those of cortical neurons. The amount of peak synaptic markers expressed was also higher in cortical neurons than that in hippocampal neurons. These results strongly suggest the usefulness of primary cultured neurons from mice embryos for synaptic function and plasticity studies, because of their clear and typical patterns of morphology that establish synapses. Results from this study also suggest the proper amount of time in vitro according to neuronal types (cortical or hippocampal) when utilized in experiments related with synaptogenesis or synaptic activities.

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 in vitro period. Unlike GAP-43, granular immunoreactive patterns of synaptophysin along the neurites were clearly found from day 2 in vitro with relatively high immunoreactive levels. Expression of synaptic markers from cortical neurons reached peak level earlier than that of hippocampal neurons, although neurite outgrowths of hippocampal neurons were faster than those of cortical neurons. Th e amount of peak synaptic markers expressed was also higher in cortical neurons than that in hippocampal neurons. Th ese results strongly suggest the usefulness of primary cultured neurons from mice embryos for synaptic function and plasticity studies, because of their clear and typical patterns of morphology that establish synapses. Results from this study also suggest the proper amount of time in vitro according to neuronal types (cortical or hippocampal) when utilized in experiments related with synaptogenesis or synaptic activities.
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 fi rst time [12]. Primary cultures off er 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 autofl uorescence, 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][14][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][18][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][26][27]. Synaptophysin is one of the synaptic vesicular proteins that has recently been characterized for the fi rst 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 neural cell culture
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 o C. 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 in vitro period. At the time of media change, only half of the media was removed and replaced with same amount of fresh media. To inhibit glial cell outgrowth, cytosine arabinoside (1 μM) was added at the moment of media change.

Immuno-fl uorescence staining (GAP-43 & Synaptophysin)
Cover slips those scheduled to be used for immune-fl uorescence staining were transferred into 4-well plates in day 2, day 4, and day 8 in vitro respectively. First, cultured neurons were fixed

Diff erential Expressions of Synaptogenic Markers in Primary Cultured Neuron
with ice-cold 70% ethanol for 10 min and washed with DPBS 3 times for 5 min. Nonspecific reactivity was blocked by adding 1% Bovine serum albumin (BSA) into the primary antibody diluting solution. We used GAP-43 (1:1,000, ab7462, ABCAM) and synaptophysin (1:1,000, ab14692, ABCAM) as primary antibody of axonal growth marker and incubated for 24 hrs in 4 o C. After the termination of primary antibody reaction, cover slips were washed with DPBS 3 times for 5 min and incubated with fluorescence tagged secondary antibody (Alexa 555, goat antirabbit IgG, A21424, Invitrogen) for 90 min in RT. Cover slips were washed again 3 times for 5 min aft er the termination of secondary antibody incubation. Finally, nuclei of cultured neurons were counter-stained with DAPI (ABBOT Molecular) and immediately investigated under laser confocal microscope system.

RESULTS
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.

Morphological changes of primary cultured neurons along the time courses
We investigated morphological changes of primary cultured neurons day by day from day 1 in vitro. Hippocampal neurons showed rather fast growing pattern so we could find definite neurite sprouting and outgrowth even from day 1 in vitro. Neurite outgrowth was faster in the areas of relatively high neural populations whereas some isolated neurons in the areas with very low neural populations showed apoptotic features (data not shown). Cultured hippocampal neurons of neighborhood gradually moved to each other showing patterns of aggregating groups and at this stage, neurites of various lengths radiated toward all directions to establish huge meshwork of neurites that were forming numerous synapses to each other (Fig. 1B).
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 in vitro (Fig. 1C). Furthermore, we could fi nd more frequent apoptotic features in case of cortical neurons than hippocampal

Yun-Gi Kim and Young-Il Lee
neurons (data not shown). Although the neurite sprouting and outgrowth of primary cultured cortical neurons were slow than those of hippocampal neurons, cortical neurons showed almost same morphological features with hippocampal neurons toward the end of second week in vitro (Fig. 1D). Neurite outgrowth of primary cultured cells stopped from the day 14 or 18 in vitro and decayed slowly thereafter in both of cortical and hippocampal neurons.

Immunofl uorescence fi ndings
GAP-43 immunoreactivities were gradually increased according to these morphological changes from day 2 to day 8 in vitro. In day 2 in vitro, GAP-43 expressions still remained relatively low and hard to find granular immunofluorescence patterns along the outgrowing neurites. GAP-43 immunoreactivities increased rapidly thereafter as neurites extend and make connections with neighboring neurons. Typical granular patterns of GAP-43 immunofluorescence along the outgrowing neurites (Fig.  2D, E) could be found from day 4 in vitro and the intensity of immunofluorescence already reached maximum during the early stages of culture (day 8 in vitro). Unlike Gap-43, granular immunoreactive patterns of synaptophysin along the outgrowing neurites (Fig. 3A, C, D) were clearly found from day 2 in vitro with relatively high immunoreactive levels (Fig. 3A). These granular findings of GAP-43 and synaptophysin immunoreactivities also resembled synaptic button like structures when considered only in morphology. Patterns of GAP-43 and synaptophysin immunoreactivities were similar in both types of cortical and hippocampal neurons. Morphological synaptic connections among the neighboring neurites could be confi rmed more clearly

Western blotting fi ndings
Along with the immunofl uorescence, quantifi cation of synaptic marker expressions was examined by western blotting through the wide range of in vitro periods. GAP-43 and synaptophysin were detected from day 4 in vitro but the expression levels between hippocampal and cortical neurons were different with the time courses. In cortical neurons, GAP-43 and synaptophysin expressions reached highest levels within 1 week (day 5) in vitro. However in hippocampal neurons, both GAP-43 and synaptophysin expressions reached highest levels after 1 week (day 10) in vitro. In cortical neurons, the amounts of GAP-43 and synaptophysin expressions were considerably higher when they reached to highest levels than those in hippocampal neurons. Th e diff erences between cortical and hippocampal neurons were more prominent in case of synaptophysin (Fig. 4B).

DISCUSSION
We characterized neurite outgrowth and synaptogenic activities in primary cultured cortical and hippocampal neurons using immunofl uorescence and Western blotting. Th e 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][35][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  Yun-Gi Kim and Young-Il Lee 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 confi rm whether they are true synaptic buttons, these fi ndings 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 clarifi ed, 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. Th ese 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 thereaft er. 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 fi ndings 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 diff erentiation 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 diff erent mechanisms according to their origin.