Primary culture of adult cortical neurons from reptile Gekko japonicus

Abstract

Adult neurons of several reptiles still retain the ability of axonal regeneration in contrast to the low intrinsic regenerative capacity of those in the central nervous system (CNS) in mammals. This feature of the reptilian neurons has provided a perfect model for elucidating the regenerative mechanism lost in the mammalian counterparts. However, little information is available on the primary culture method of adult reptilian neurons, which greatly limits their valuable applications. In the present study, we introduced a simple and easy method for the isolation, culture, and identification of neurons from the cerebral cortex using the adult geckos. The cultured cells were further identified by immunofluorescence using antibodies against neuron‐specific markers β‐Ⅲ‐tubulin and NeuN. The cortical neurons from adult gecko displayed spindle‐shaped, bipolar, or multipolar morphology with a plump soma. This primary culture method for adult reptilian neurons will be beneficial for comparative studies of neuronal biology in various vertebrates.

The primary gecko neurons express neuronal linage markers of mammals.

1. INTRODUCTION

Injury to the central nervous system (CNS) in adult mammals leads to permanent functional deficits due to limited regenerative capability of the neurons and regulation by glial and immune cells (Chen et al., 2017). Unlike mammals, several other vertebrates including fish, amphibians, and reptiles have the ability to regrow axons and repair functional circuits (Diaz Quiroz & Echeverri, 2013; Rasmussen & Sagasti, 2017; Sun et al., 2018; Szarek et al., 2016). For instance, during the regeneration of the spinal cords in these animals, the existing or newly generated neurons grow axons along the channels formed by ependymal glial cells in a permissive regenerative milieu without evoking serious inflammation and glial scar (Diaz Quiroz & Echeverri, 2013). The neurons in the CNS of the regenerative models exhibit robust survival and regenerative capacity following injury, in stark contrast to those of the mammals. Such exclusive features are governed by a different set of molecular programs with the non‐regenerative counterparts (Lee‐Liu et al., 2013). Several established methods, such as transgenic tracing, single cell sequencing, and immunostaining, can elucidate the states of in vivo axonal growth or gene expression profiles (Kunst et al., 2019; Tambalo et al., 2020; Tiklova et al., 2019), but an in vitro culture of primary neurons from the regenerative models is indispensable for investigating neuronal reactions to various stimuli and the basic mechanisms of differential functions.

Some reliable approaches for primary culture of the CNS neurons have been successfully established using the fetal spinal cord or brain tissues in fish, frogs, chickens, mice, and rats (Chen et al., 2013; Kumar & Mallick, 2016; Lohof et al., 1992; Lumsden & Davies, 1986; Tessier‐Lavigne et al., 1988). However, in vitro culture of neurons from the adult CNS tissues is not universal because of the requirement of more delicate culture conditions (Eide & McMurray, 2005). It has been shown that the fetal CNS neurons are physiologically immature, leading to limitations in mechanistic studies on regenerative capacity lacking in adult counterparts, as well as age‐related cellular events occurring in the adult neurodegenerative diseases, including Parkinson's disease (PD), Alzheimer's disease (AD), and prion disease (Majd et al., 2008; Slanzi et al., 2020). Moreover, fetal CNS neurons lack receptor systems as present in the adult neurons (Brustein et al., 2003; McLean & Fetcho, 2004). Therefore, in vitro preparation of CNS neurons from adult regenerative models is experimentally more relevant for elucidating regeneration of neuronal axons.

Several amniotic reptiles such as lizards and turtles still retain the ability for the anatomical and functional recovery of the lumbar and thoracic cord following complete transection (Alibardi, 2020; Rehermann et al., 2009). The neurons isolated from the adult reptiles are phylogenetically closer to those in the mammals, and will be advantageous in identifying key factors that orchestrate axonal regeneration in the presence of various stimuli. To the best of our knowledge, no such methodology has been described yet. Therefore, in the present study, we developed a method for the primary culture of neurons from the cerebral cortex of adult gecko. The cultured neurons displayed both spindle bipolar and multipolar morphology with >95% purity, as evidenced by β‐Ⅲ‐tubulin and NeuN immunostaining. The methodology will be beneficial for the study of animal CNS regeneration.

2. MATERIALS AND METHODS

2.1. Animals

Adult Gekko japonicus geckos were obtained from the Experimental Animal Center of Nantong University. They were fed mealworms ad libitum and were housed in an air‐conditioned room with a controlled temperature (22–25°C) and saturated humidity.

All animal experiments were conducted in accordance with the guidelines of the NIH (Guide for the Care and Use of Laboratory Animals: 1985) and the Guidelines for the Use of Animals in Neuroscience Research by the Society for Neuroscience. The experiments were approved by the Animal Care and Use Committee of Nantong University and the Jiangsu Province Animal Care Ethics Committee.

2.2. Primary cell culture of the adult gecko cerebral cortex neurons

The geckos were euthanized on ice and sterilized with 75% alcohol before dissection of the entire brain. The brain was placed in a precooled anatomical solution containing 1:1 Dulbecco's modified Eagle's medium/Ham's F‐12 (Sigma, 56498C) medium and 1% penicillin/streptomycin (P/S, Beyotime, C0222, 1:100). Furthermore, the cerebral cortex was separated under an anatomical microscope, and the surrounding vessels were removed by immersing samples in a 5‐ml centrifuge tube containing 3 ml of anatomical solution. The cortex tissues in the precooled anatomical solution were centrifuged at 168 g for 1 min at three times. Furthermore, they were digested with 1 ml of 0.2% collagenase (Solarbio, C8140) for 15–18 min in an incubator with shaking every 5 min, followed by digestion with 4 ml of 0.25% trypsin (Gibco, 25200072) for 20–25 min at 30℃ with shaking every 5 min. The cell suspension was obtained by adding an equal amount of DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS, Gibco, 10099133C) and 1% P/S to stop the dissociation. Next, the suspension was filtered through a 400 mesh sieve. The filtered solution was collected and centrifuged at 168 g for 10 min, and the supernatant was discarded. The samples were further mixed with DMEM/F12 medium supplemented with 10% FBS and 1% P/S to resuspend the cells, and cells at a density of 2 × 10⁶ cells/ml were plated into culture dishes (35 × 10 mm) coated with PDL (Sigma, P6407). They were cultured in a humidified atmosphere containing 5% CO₂/95% air for 24 h at 30°C, followed by culturing in Neurobasal medium (Gibco, 21103049) supplemented with B27 (2% STEMCEL Technologies, 05711), P/S (1%), NGF (0.5 μg/ml, RDSYSTEMS, 556‐NG), and glutamine (1%, Beyotime, C0212). The medium was changed every 3 days until days 7–9.

2.3. Cell immunofluorescence staining

After 7 days of culture, the Neurobasal medium was discarded and the cells were washed with phosphate‐buffered saline (PBS, pH 7.4, Corning, 21‐040‐CM). Furthermore, cells were then fixed with 4% paraformaldehyde at room temperature for 45 min, followed by washing with PBS twice. After incubation with blocking buffer containing 3% BSA (BioFroxx, 4240GR100), 0.1% Triton X‐100 (Sigma, 93443), and 10% normal goat serum (Bioss, C‐0005) at 37℃ for 1 h, the cells were further incubated with primary antibodies against β‐Ⅲ‐tubulin (1:1000, BioLegend, 801201), NeuN (1:400, Abcam, ab177487), GFAP (1:1000, Abcam, ab7260), or MBP (1:1000, Abcam, ab218011) overnight at 4℃. The fixed cells were washed with PBS three times for 10 min each, and subsequently incubated with a second fluorescent Cy3‐labeled goat anti‐rabbit (1:400, Proteintech, SA00009‐2) or the Alexa Fluor 488‐labeled donkey anti‐mouse IgG (1:400, Abcam, ab150105) at 4℃ overnight. After washing with PBS three times for 10 min each, the cells were stained with Hoechst 33342 (1:4000, Sigma, 14533) for 15 min at room temperature before being observing under a fluorescence microscope (Leica).

2.4. Statistical analysis

The statistical significance of the differences between groups was analyzed by one‐way analysis of variance (ANOVA) followed by Bonferroni's post hoc comparison test using SPSS version 15.0 (SPSS). The normality and homoscedasticity of the data were verified prior to statistical analysis using Levene's test. p < 0.05 and p < 0.01 were considered to be statistically significant.

3. RESULTS

3.1. Morphology of primary cortical neurons isolated from lower amniotic geckos

To obtain primary cortical neurons, the cerebral cortex of adult geckos was dissected on ice, and subsequently digested for neuronal culture. The isolated cells were successfully cultured on PDL‐coated plates as a monolayer, with a uniform spherical morphology (Figure 1a). When cultured for 1 day, most of the cells adhered to the bottom of the dish without outgrowth of neurites (Figure 1b). However, by day 3 of in vitro (DIV3) culture, some cells showed visible sprouting of neurites, with the longest one potentially assigned to the axon (Figure 1c). At DIV5, the growing axons of the cells were significantly distinct, and their neurites began to form connections with those of other cells (Figure 1d). At DIV7, the length of cell neurites continued to extend, with an appearance of bipolar or multipolar morphology (Figure 1e).

FIGURE 1.

Morphological photographs of the gecko primary neurons cultured at different times. (a) Flow chart of the procedure for culturing the cortical neurons from adult gecko. (b) Primary neurons adhered to the dish bottom without growth of neurites at day 1 of in vitro (DIV1) culture. (c) At DIV 3, several cells showed visible sprouting of neurites with long axons. (d) Neurites of the neurons at DIV5 developed connection with those of others. (e) At DIV 7, the length of cell neurites continued to extend, with appearance of bipolar or multipolar morphology. Arrows indicate axons, and arrowheads indicate connection of neurites. Scale bar = 50 µm

3.2. Primary gecko neurons express neuronal linage markers of mammals

To understand whether primary neurons from reptiles express similar neuron‐specific markers similar to those from mammals, we examined the primary gecko neurons using the NeuN and β‐Ⅲ‐tubulin antibodies that stain the neuronal nucleus, and cytoplasm and neurites of neurons, respectively (Lee et al., 1990). The analysis suggested that the nucleus was NeuN‐positive, whereas the cytoplasm and neurites were β‐Ⅲ‐tubulin‐positive signals in the primary neurons (Figure 2a–d). These data indicate that the primary gecko neurons express neuronal lineage markers similar to those in mammals.

FIGURE 2.

Immunofluorescence staining of gecko primary neurons with neuron‐specific antibodies. The neurons are stained with (a) β‐Ⅲ‐tubulin, (b) NeuN antibodies, and (c) Hoechst 33342 after culture for 7 days. (d) The merge of (a–c). Scale bar = 50 µm

3.3. Purity determination of primary cultured neurons

To ascertain the neuronal purity and success of the isolation procedure, we performed immunofluorescence using antibodies against β‐Ⅲ‐tubulin, GFAP, and MBP to quantify the percentage of neurons, astrocytes, and oligodendrocytes, respectively, in the primary culture. The analyses detected no GFAP‐positive astrocytes or MBP‐positive oligodendrocytes in the culture (Figure 3a–c). In contrast, all neurons were stained with β‐Ⅲ‐tubulin antibody (Figure 3d–f). The data indicate that the primary neurons isolated from the gecko cerebral cortex have reached a high purity required for various experimental analyses.

FIGURE 3.

Purity determination of gecko primary neurons. The cells are immunostained with antibodies against (a) GFAP, (b) MBP, or (d) β‐Ⅲ‐tubulin after culturing for 7 days. (c and e) Hoechst 33342 is used to stain the cell nucleus. (f) The merge of (d) and (e). Scale bar = 50 µm

3.4. NGF efficiently promotes neurite extension in the gecko primary cortical neurons

As NGF regulates the growth, survival, and developmental plasticity in neurons in the vertebrates (Rocco et al., 2018), we challenged the primary neurons from gecko with NGF (0.5 μg/ml) for 24 h to observe its effects on neurite extension. Incubation with NGF significantly promoted branching and extension in neurites of the gecko primary cortical neurons, consistent with our hypothesis (Figure 4a–c). Therefore, the data indicate that the nerve growth factors are efficient in promoting neurite growth in the gecko primary neurons.

FIGURE 4.

Effects of NGF on the growth of neurites in gecko primary neurons. The cells are cultured in Neurobasal medium in the absence (a) or presence (b) of 0.5 μg/ml of NGF for 7 days. (c) and (d) are statistics of (a) and (b), respectively. Scale bar = 50 µm. Data are expressed as the mean ± SD, *p < 0.05 was considered to be statistically significant

4. DISCUSSION

Primary neurons are cultured as a tool for understanding neuronal biology under defined conditions, as an alternative to in vivo systems. However, it is comparatively more difficult to isolate viable neurons from adult vertebrates (Eide & McMurray, 2005). Nevertheless, the application of adult neurons in deciphering neurophysiology has unique advantages over the immature embryonic preparations, such as in electrophysiological analyses (Meade et al., 2019). The methodology for culturing adult neurons has been established in several vertebrates, including fish and mammals (Eide & McMurray, 2005; Meade et al., 2019), but, to the best of our knowledge, the preparation of neurons from adult reptiles has not been documented yet. Thus, in the present study, we introduced a reliable approach for the isolation and culturing of cortical neurons from adult gecko making them available for the study of differential responses to various stimuli and mechanisms of axonal regeneration in adult.

Our methodology can maintain the culture of adult gecko neurons for up to 9 days or more in vitro, in a humidified chamber at 30°C in 5% CO₂/95% air. This period is sufficient for studies of cell events and the cell–cell interactions in controlled environments. Although digestion with trypsin and collagenase which may lead to poor cell viability (Majd et al., 2008; Wu et al., 2009), the system is still efficient in supporting cell adherence and neurite outgrowth of the isolated neurons by using mild 0.2% collagenase. Moreover, the optimal temperature for culturing reptilian neurons was found to be optimal at 30°C (Milton et al., 2007), which is completely different from that for mammalian cells. This may be derived from the adaptive evolution of poikilotherms in the phylogeny.

Previous studies have demonstrated that primary neurons from the juvenile turtle Trachemys scripta displayed NeuN‐ and GFAP‐positive staining (Milton et al., 2007). GFAP is expressed not only by mature astrocytes but also by neural stem cells (Wang et al., 2021). Therefore, it can be postulated that primary neurons from juvenile reptiles retain cell stemness. In contrast, primary neurons from the adult gecko cerebral cortex exhibit NeuN‐positive, but not GFAP‐positive signals, suggesting isolation of fully differentiated mature neurons that may help in appropriate applications.

5. CONCLUSIONS

In conclusion, we successfully established a system for primary culture of neurons from adult geckos. The neurons exhibited NeuN‐positive and β‐Ⅲ‐tubulin‐positive signals with bipolar or multipolar morphology. Moreover, the primary neurons showed a robust reaction to stimulation by NGF. Therefore, this methodology will be beneficial for studying the neuronal biology of reptiles.

CONFLICT OF INTEREST

The authors have declared that no competing interests exist.

AUTHOR CONTRIBUTIONS

Yjie W and Yjun W designed the study and wrote the manuscript. BH performed the experiments. BH, CS, HL, TY, and HS analyzed the data. Yjun W participated in the discussion. All of the authors have approved the present version of the manuscript and have agreed to be accountable for all aspects of the work regarding questions related to the accuracy or integrity of any part of the work.