Exp Neurobiol 2017; 26(3): 158-167
Published online June 30, 2017
© The Korean Society for Brain and Neural Sciences
Junsung Woo1,2,3†, Sun-Kyoung Im4†, Heejung Chun1,2, Soon-Young Jung2, Soo-Jin Oh1,2,3, Nakwon Choi5,6, C. Justin Lee1,2,3,7* and Eun-Mi Hur1,4,7*
1Center for Neuroscience, Korea Institute of Science and Technology (KIST), Seoul 02792, 2Functional Connectomics, Korea Institute of Science and Technology (KIST), Seoul 02792, 3Center for Glia-Neuron Interaction, Korea Institute of Science and Technology (KIST), Seoul 02792, 4Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, Korea Institute of Science and Technology (KIST), Seoul 02792, 5Center for BioMicrosystems, Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul 02792, 6Division of Bio-Medical Science &Technology, KIST School, Korea University of Science and Technology, Seoul 02792, 7Department of Neuroscience, Division of Bio-Medical Science &Technology, KIST School, Korea University of Science and Technology, Seoul 02792, Korea
Correspondence to: *To whom correspondence should be addressed.
C. Justin Lee, TEL: 82-2-958-6940, FAX: 82-2-958-6937, e-mail: firstname.lastname@example.org
Eun-Mi Hur, TEL: 82-2-958-6856, FAX: 82-2-958-6937, e-mail: email@example.com
†These authors contributed equally
Brain is a rich environment where neurons and glia interact with neighboring cells as well as extracellular matrix in three-dimensional (3D) space. Astrocytes, which are the most abundant cells in the mammalian brain, reside in 3D space and extend highly branched processes that form microdomains and contact synapses. It has been suggested that astrocytes cultured in 3D might be maintained in a less reactive state as compared to those growing in a traditional, two-dimensional (2D) monolayer culture. However, the functional characterization of the astrocytes in 3D culture has been lacking. Here we cocultured neurons and astrocytes in 3D and examined the morphological, molecular biological, and electrophysiological properties of the 3D-cultured hippocampal astrocytes. In our 3D neuron-astrocyte coculture, astrocytes showed a typical morphology of a small soma with many branches and exhibited a unique membrane property of passive conductance, more closely resembling their native
Keywords: 3D culture, reactive astrocyte, adenovirus, functional characterization, passive conductance, tonic GABA current
Astrocytes are the most abundant cells in the mamamlian central nervous system and are recognized as important regulators of brain function [12,13]. Astrocytes become reactive in a number of pathological conditions, following injury and in many neurological disorders, such as Alzheimer's and Parkinson's diseases [14,15,16,17], to name a few. Although the concept of reactive astrogliosis and its molecular definition is still incomplete, astrocytes are considered reactive when they become hypertrophic and increase the expression of molecular markers for reactive astrocytes, such as GFAP (glial fibrillary acidic protein), nestin, and vimentin [14,15,17,18], Recently, increases in GABA content and tonic GABA release in astrocytes have also been suggested to indicate astrogliosis, especially in neurodegenerative diseases [16,19].
Developing a physiologically relevant 3D culture system is of particular importance for astrocyte studies, because it is well known that in a conventional 2D culture system, astrocytes do not preserve many of the structural and functional features observed
The aim of this study was to develop a physiologically relevant culture system that preserves important features of both resting and reactive astrocytes. By coculturing neurons and astrocytes in 3D and analyzing the morphological, molecular biological, and electrophysiological properties of the 3D-cultured astrocytes, here we show that astrocytes grown in 3D collagen matrix more closely resemble their
Pregnant ICR mice (E18.5) were purchased from DBL (Eumseong, Korea) and sacrificed for primary culture of neurons and glia, following a previously described protocol with minor modifications . Briefly, embryos were decapitated and the entire hippocampi were dissected out and were treated with papain (Worthington) and serially triturated. Dissociated cells were counted and seeded at a density of 1×105 cells ml-1 for 2D and 4×106 cells ml-1 in a collagen mixture for 3D. Cells were cultured in plating medium consisting of neurobasal media supplemented with 5% fetal bovine serum (GIBCO), 2% B27-supplement (Invitrogen), 2 mM Glutamax-I (GIBCO) and 1% penicillin-streptomycin (GIBCO). After 1 day, plating medium was replaced by serum-free medium and maintained at 37℃ in a 5% CO2 humidified incubator. One-half of the medium was replaced with fresh culture medium every 2~4 days. Cultured cells growing in 500 µl media were treated with 1 µl adeno-CMV-mCherry virus (viral stock titer: 4.5×1012 genome copies (GC) ml-1) for 4 days.
Commercial type I collagen (8~11 mg ml-1 in acetic acid (0.1% [w/v] for custom-extracted collagen and 0.02 N for commercial collagen), rat tail; Corning or custom-extracted) was used and neutralized (pH 7.5) collagen solutions were prepared as reported previously . Total of 500 µl of a 2.5 mg ml-1 collagen solution was prepared by adding 110~160 µl collagen stock (depending on the concentration of collagen stock solution), 50 µl of 10x Dulbecco Modified Eagle Medium (DMEM; Sigma-Aldrich). 10~20 µl of 0.5 N NaOH for neutralization, and 50 µl of cell-suspension in DMEM. Final volume of 500 µl was matched by adding 1x DMEM (Lonza). Cells were seeded in collagen at a density of 4×106 cell ml-1. All solutions were added on ice to minimize undesired gelation. Collagen seeded with cells was then immediately processed for gelation (37℃, 30 min), and media was added.
To stain neurons and astrocytes, 3D constructs were fixed in 4% [w/v] paraformaldehyde and blocking was performed with 2% [w/v] BSA in PBS containing 0.1% [w/v] Triton X-100 for 2 hr. 3D constructs were then incubated sequentially with primary and secondary antibodies diluted in the blocking solution at 4℃ overnight. Following primary antibodies were used: mouse anti-βIII-tubulin (TuJ1) (1:1,000; Abcam), chicken anti-GFAP (1:500; Millipore). Alexa Fluor conjugates (Alexa Fluor 488 and 594) (1:1,000; Molecular Probes) were used for secondary antibodies. Nuclei were stained with Hoechst 33342 (1: 5,000; Molecular Probes). All samples were rinsed with PBS between the incubation steps.
Fluorescence images were acquired using an inverted confocal laser scanning microscope (LSM 700; Carl Zeiss) equipped with solid-state lasers (405, 488 and 555 nm). Post-image processing such as maximum intensity projection and 3D reconstruction was performed using ZEN 2012 software (Carl Zeiss). Z-stacked images (stack size, 17~176 µm; step size, 0.94~3.4 µm) were acquired with 10x and 20x objectives.
Electrophysiological recordings were performed with co-culture of hippocampal neurons and astrocytes at DIV (days
Mice (8~10 weeks old, C57BL/6 background) were anesthetized by intraperitoneal injection of 2% avertin (20 µl g-1) and placed into stereotaxic frames. Adenovirus containing mCherry was loaded into a micro dispenser (VWR, USA) and injected bilaterally into the hippocampal dentate gyrus (DG) region at a rate of 0.3 µl min-1 (total 2 µl) with a 25 µl syringe using a syringe pump KD Scientific, USA). The stereotaxic coordinates of the injection site were 1.7 mm away from the bregma and the depth was 2 mm beneath the skull. All experimental procedures described below were conducted according to the animal welfare guidelines approved by Institutional Animal Care and Use Committee of the Korea Institute of Science and Technology.
The significance of data for comparison was assessed by Student's two-tailed unpaired t-test between two groups and one-way ANOVA test between three groups. Analyses were performed with Prism (GraphPad Software, Inc.) and Clampfit software. The data distribution was assumed to be normal. Data are presented as mean±SEM (standard error of the mean). Levels of statistical significance are indicated as follows: *(p<0.05), **(p<0.01), ***(p<0.001).
To assess the morphology of cells in the 3D neuron-astrocyte co-culture, we performed immunocytochemistry by using antibodies against Tuj1, a neuron-specific class III β tubulin, and GFAP, an astrocytic filament protein. In striking contrast to neurons that show similar morphology in 2D and 3D cultures, astrocytes in the two cultures exhibited a remarkable difference. In the standard 2D culture, astrocytes had a flat, polygonal morphology, which is very different from their
High-titer adenoviral transduction of astrocytes has been shown to cause local and selective virus-induced reactive astrocytosis
We next compared morphological and biochemical characteristics of reactive astrocytes in adenovirus-infected hippocampal brain slice with those in our 3D cell culture system. Similar to our 3D culture (see Fig. 1, 2, 3A and 3B), we found cellular hypertrophy and significant increase of GABA signal in reactive astrocytes from the DG region of hippocampal brain slice infected with adenovirus (Fig. 3C and 3D). These
The increased intracellular GABA content in reactive astrocytes can be released tonically from astrocytes and detected in neighboring neurons in the form of tonic inhibition current [16,19]. To test this in 3D culture at DIV 7, we measured the tonic GABA current using the GABAA antagonist bicuculline from a neuron in the vicinity of viral infection (Fig. 4A). We found a significant increase in tonic GABA current in adenovirus treated condition compared to control condition (Fig. 4A, B), indicating an increase in tonic GABA release from reactive astrocytes. It has been suggested that astrocytes synthesize GABA through monoamine oxidase B (MAO-B) pathway with putrescine as an initial substrate [16,23,24]. To test if putrescine pathway is intact in our 3D culture system, we measured the tonic GABA current in normal 3D-cultured astrocytes treated with putrescine. We found a significant enhancement of tonic GABA current after putrescine treatment (Fig. 4C). We found a very similar trend in the DIV 14 culture, compared to DIV 7 condition, except that the magnitude of putrescine- and adenovirus-induced tonic GABA current was much higher at DIV 14 (Fig. 5A~D). These results suggest that tonic GABA release from reactive astrocytes is detected in adenovirus-infected reactive astrocytes.
Fully differentiated, mature astrocytes have been shown to display a leaky membrane property with extremely low membrane resistance (ranging from 1~10 MΩ), namely a passive conductance with a linear current-voltage relationship . It has been recently demonstrated that the most of passive conductance is mediated by the heterodimer of two-pore potassium (K2P) channel subunits, TREK-1 and TWIK-1 . Therefore, the presence of passive conductance can serve as a useful electrophysiological marker of astrocytes. To test whether our 3D-cultured astrocytes display this electrophysiological property, we measured the passive conductance from individual astrocytes by whole-cell patch-clamping. We found that the passive conductance was readily observed in 3D-cultured normal and reactive astrocytes (Fig. 4E-G, 5E-G). Interestingly, passive conductance was significantly increased in reactive astrocytes at DIV 14 (Fig. 5G), but not at DIV 7 (Fig. 4G). This result is consistent with previous reports that showed an enhancement of passive conductance in ischemic condition accompanying reactive astrocytes due to an increase in K2P channel expression [27,28]. Taken together, these results suggest that 3D-cultured reactive astrocytes display the electrophysiological features as evidenced by the increased tonic GABA release and passive conductance, resembling pathophysiological conditions
Here we developed a collagen-based 3D coculture system composed of neurons and astrocytes and examined the morphological, molecular biological and electrophysiological properties. In basal condition, 3D-cultured astrocytes exhibited a distinct stellate morphology with a small soma and several branches and showed passive conductance, resembling resting astrocytes observed
Biological functions of some types of cells are particularly difficult to study
Together with previous findings [6,11], this study further confirms the advantages and appropriateness of the 3D culture system for studying astrocytes. By minimizing baseline reactivity, it is possible to induce astrogliosis applying a whole range of experimental approaches. As an example, here we infected the culture with high-titer adenovirus and observed morphological and functional changes in astrocytes, in a way that mimics astrogliosis observed in
In summary, 3D coculture of neurons and astrocytes can be adapted to reconstruct normal and diseased conditions, thereby helping us to gain further insights towards understanding the multifaceted and complex roles of astrocytes.