Cell-to-Cell Communication

Theofylaktos Apostolou
and Spyridon Kintzios

 

Evidence of Near-Instantaneous
Distant, Non-Chemical Communication between Neuronal (Human SK-N-SH Neuroblastoma) Cells by Using a Novel Bioelectric Biosensor

Abstract: According to an increasing number of reports, non-chemical, distant cellular interactions (NCDCI) may be responsible for a yet underestimated mechanism of cell-to-cell communication and coordinated cellular responses. Based on these and other experiments, an electromagnetic nature of consciousness has been proposed (e.g. conscious electromagnetic information (CEMI) field theory). In the present study, we provide supporting evidence for this theory by applying a novel bioelectric biosensor in order to simultaneously investigate changes of the membrane potential of physically separated human SK-N-SH neuroblastoma cells. We demonstrate the existence of distant communication between neuroblastoma cells. When one of the isolated groups of cells (‘inducer’) was stimulated with the neuro­transmitter dopamine, a synchronized response was observed in the neighbouring but physically separated cell group (‘detector’). The range of this phenomenon was decreased with increasing distance. In the absence of a dopamine-induced stimulus, no clear recognizable pattern of synchronized response was observed. The nature of mechanisms underlying the observed distant cellular interactions is discussed in view of the observed coordinated patterns of changes of the cell membrane potential.

Keywords: Bioelectric Recognition Assay (BERA); biosensor; dopa­mine; non-chemical; distant cell interactions (NCDCI); neuroblastoma

1. Introduction

During the past two decades, several experiments have provided strong indications for the existence of a distant, non-chemical communication between cells or cell populations not having direct contact to each other (Wainwright, 1998; Nikolaev, 2000; Trushin, 2003; 2004; Fels, 2009; Cifra, Fields and Farhadi, 2011). The terms non-chemical, non-electrical (NCNE) signalling and, more recently, non-chemical, distant cellular interactions (NCDCI) have been pro­posed to describe such phenomena (Wainwright, 1998; Farhadi, 2014). A landmark study in the field was reported by Farhadi et al. (2007), who demonstrated that treatment of Caco-2 cell cultures (termed the ‘inducer’) with H2O2 could be sensed by similar cultures (the ‘detector’) placed in separate containers to the inducer but not exposed to H2O2. This kind of synchronized response was expressed as a similar pattern of reduced total protein content and increased nuclear NFκB activation and cytoskeletal damage in both inducer and detector cells. In another study, Chaban et al. (2013) provided evidence that dorsal root ganglion (DRG) neurons could sense the presence of neighbouring, physically disconnected apoptotic DRG or human neuroblastoma SH-SY5Y cells by considerably altered ATP- and capsaicin-mediated [Ca2+]і influx. Hashemibeni et al. (2014) reported the detection of NCDCI among adipose-derived stem cells, expressed as changes in cell proliferation presumably due to the vicinity of the inducer cells (stem cells treated with fibroblast growth factor). More recently, Fels (2016) demonstrated the effect of physically separated cultures of autotrophic unicellular species Euglena viridis on the proliferation rate of the heterotrophic uni­cellular species Paramecium caudatum and the multicellular hetero­trophic Rotatoria sp.

Various mechanisms have been suggested in order to explain the reported signalling events between distant cells and tissues. The majority of the observed cases have been attributed to electromagnetic radiation (Cifra, Fields and Farhadi, 2011; Reguera, 2011), particu­larly biophoton emission from 200 to 800 nm as a result of micro­tubule oscillations and/or free radical species production (Prasad et al., 2014; Tang and Dai, 2014; Pospíšil, Prasad and Rác, 2014; Craddock et al., 2012). In particular, advanced molecular simulation experiments have highlighted the intricate relationship between reactive oxygen species (ROS) and the phosphorylation state of microtubule-associate proteins such as tau, rich in aromatic amino acids, especially in respect of mediating UV light-based information through processes of quantum coherent energy transfer (Craddock et al., 2014; Kurian, Obiesan and Craddock, 2017). It should be mentioned, however, that the physical feasibility of the electro­magnetic cell-to-cell communication has been challenged, especially in view of the doubtful ability of single cells to spatially process electromagnetically-mediated information, as well as the very low impact of such radiation on biological systems (Kučera and Cifra, 2013). The occurrence of a distant, chemical in nature, cellular inter­action due to the exchange of volatile molecules has been hypo­thetically formulated, though not proven (Hashemibeni et al., 2014). The possible role of sound waves has also been demonstrated in the distant communication between bacteria (Matsuhashi et al., 1998).

Verification of electromagnetic interaction between neural cells would contribute considerably to mounting evidence of the existence of a neuroelectromagnetic field able to interact with the brain to form a unified consciousness field (Jones, 2017).

In the present study we investigated the occurrence of NCDCI between human SK-N-SH neuroblastoma cells by using a novel bio­sensor that allowed for the simultaneous, real-time monitoring of bio­electric properties of physically separated inducer and detector cells. We demonstrate that changes of the membrane potential caused by the neurotransmitter dopamine (in inducer cells) can be almost instantly detected by neighbouring cells (detector cells), which are not exposed to dopamine and are physically isolated from inducer cells, therefore excluding the exchange of chemical signals. In the absence of a dopamine-induced stimulus, no clear recognizable pattern of synchro­nized response was observed.

2. Materials and Methods

2.1. Chemicals

Human neuroblastoma SK-N-SH cell cultures were originally pro­vided from LGC Promochem (UK) and subcultured in Dulbecco’s medium with 10% fetal bovine serum (FBS), 1U μg-1 antibiotics (penicillin/streptomycin) and 2 mM L-glutamine which were provided from Invitrogen (CA, USA). All other reagents were purchased from Sigma-Aldrich (Taufkirchen, Germany).

2.2. Cell membrane potential measurements: biosensor set-up

Changes of the cell membrane potential were measured using a specially designed multichannel potentiometer (Uniscan, Buxton, UK), operating according to the principle of the Bioelectric Recog­nition Assay (Kintzios et al., 2001; Ferentinos et al., 2013). The system is able to provide real-time measurements of changes of the electric properties of neural cells in response to various neurotrans­mitter agonists and/or antagonists (Apostolou et al., 2017). It allows for up to eight simultaneous measurements from respective positions (Positions 1–8), each comprising a pair of screen-printed electrodes (working electrode: carbon, reference: Ag/AgCl) on a disposable sensor strip (DropSens, Asturias, Spain). The distance between adjacent measuring positions is 3.7 mm. In this array of elec­trodes and respective measuring channels, inducer cells (SK-N-SH cells treated with dopamine) were always placed on Position 1, while detector cells (non-treated SK-N-SH cells) were placed on either Position 2, 3, or 4. In this way, it was possible to investi­gate the effect of physical distance between the inducer and the detector cells on the range of the NCDCI signalling.

Before each assay, cells were detached from the culture and con­centrated by centrifugation (2 min, 1200 rpm, 25°C). During each assay, cells in suspension were placed on the top of the designated Positions on the sensor strip (45 μL ≈ 50 x 103 cells) with the help of an automatic pipette. Next, 5 μL of neurotransmitter (100 μM dopa­mine) solution were added.

Immediately after the addition of dopamine in the inducer cell suspension, the response of both inducer and detector cells was recorded as a time-series of potentiometric measurements (in Volts). The duration of each measurement was 180 sec and 360 values/sample were recorded at a sampling rate of 2 Hz.

The following two experiments were conducted in the framework of the present study:

In the first experiment, inducer cells were stimulated with dopa­mine, as described above, and the response of both inducer (Position 1) and detector cells, located in either Position 2, 3, or 4 (three differ­ent experimental configurations), was recorded.

In the second experiment, the same procedure was applied but no dopamine was added in the inducer cell suspension. This was done in order to investigate the NCDCI between cells in the absence of an inducer cell population.

2.3. Experimental design

Experiments were set up in a completely randomized design, with a set of two individual replications for each inducer–detector cell con­figuration (n = 360 per replication). Each experiment was repeated five times at independent dates. Data were normalized in order to allow the comparison of measurements conducted on different dates. Differences between time-series of potentiometric measurements recorded in different positions were statistically assessed using a t-test assuming unequal variances.

3. Results

3.1. Synchronization of responses between inducer and detector cells depends on distance

The novel biosensor system allowed for the simultaneous recording of changes of the membrane potential of both the inducer cells (Position 1) and detector cells located either on an adjacent measurement channel (Position 2) or on more remote ones (Positions 3 and 4). In this way, we were able to directly compare the observed patterns of response between inducer and detector cells. The experimental set-up allowed us to ascertain that both cell populations were measured under identical experimental conditions during each experiment, with the single difference of exposing inducer cells to dopamine. It should be noted that responses from all eight (1–8) electrode positions were simultaneously recorded; however, no responses at all were observed from cell-free positions, such as Positions 5–8. Responses on Positions 2, 3, or 4 were observed only if these positions were covered with detector cells, as described in the experimental design (2.2). In other words, a measureable signal was produced only in the presence of cells on the measuring positions.

In the first experiment, an almost identical pattern of response was observed between Position 1, containing inducer cells (SK-N-SH cells treated with 100 μΜ dopamine) and the adjacent Position 2, contain­ing detector cells (SK-N-SH cells not exposed to dopamine). This was demonstrated by the essentially parallel, time-dependent patterns of cell membrane potential in both cell groups. In addition, the average, over all measurements, response of detector cells on Position 2 was slightly lower than that of inducer cells, in a statistically non-significant manner.

On the contrary, the responses of detector cells in the more distant Positions 3 and 4 were much less synchronized with inducer cells (Position 1), as revealed both by the different time-dependent patterns of cell membrane potential and the considerably lower, statistically different responses of the detector cells in these positions compared to the inducer cells.

The effect of distance on the synchronization of response is also presented in Figure 4 (solid line), where the relative differences of responses between inducer cells (Position 1) and detector cells in different positions (Positions 2, 3, or 4) are compared. The data pre­sented in the figure clearly demonstrate that the difference in response relative to the inducer cells is increased with the distance from them (from 19% in Position 2, to 43% in Position 3, and 49% in Position 4).

We have also investigated the possibility of the elicitation of a response on a cell-free electrode adjacent to the inducer cell position (Position 1). Absolutely no response was observed in this case, which is in accordance with results from different applications using the same cell-based biosensor configuration (e.g. Apostolou et al., 2017). Simply put, cell-free electrode positions could not per se sense bio­electric changes in adjacent, cell-covered positions.

Application of dopamine on cell-free positions caused an extremely low, close to zero negative response (= 0.07 ± 0.002 V) which was the same for every position recorded, i.e. both for the inducing (Position 1) and the detector (Positions 2–4) electrodes, irrespective of their experimental configuration. It was concluded, therefore, that only SK-N-SH cells contributed to the observed results. In other words, only cells in one position could trigger an effect in adjacent positions, i.e. cells were required as mediators of the distant communication mechanism.

3.2. Synchronization of responses between distant cell populations depends on dopamine-induced cell excitation

In the second experiment, no dopamine was added to the ‘inducer’ cells (Position 1), i.e. inducer cells were functionally identical to detector ones. In this case, no recognizable pattern of differences in responses appears. Differences in the response of detector cells relative to Position 1 range from 46% (Position 2) to 15% (Position 3) to 27% (Position 4) (Figure 4, dotted line). In other words, in the absence of a dopamine-induced stimulus, no clear recognizable pattern of NCDCI is observed.

4. Discussion

This is the first study regarding the investigation of non-chemical, distant communication between human neuronal cells of the same type, using, in addition, dopamine-induced neuron firing as the method to determine the inducer cell population. In this way, the chosen experimental set-up allowed for a first assessment of the possible contribution of NCDCI to the coordinated responses of neuronal cell populations following stimulation by a common neurotransmitter.

The novel, BERA-type biosensor set-up used in the present study offered two important advantages for studying the possible occurrence of NCDI between neuroblastoma SK-N-SH cells. First, it allowed for monitoring synchronization of cell responses between inducer and detector cells, expressed as changes in the cell membrane potential, at the moment these took place, i.e. in real time. This is a considerable advance compared to previous reports (for example, Farhadi et al., 2007; Chaban et al., 2013; Hashemibeni et al., 2014; Fels, 2016) where the occurrence of distant communication was evaluated by means of a series of biochemical assays such as measurement of cell proliferation (hours to days after the assumed NCDCI), total protein content, assay of NFκB activity and [Ca2+]i, as well as assessment of cytoskeletal/tight junction structure. In this way, any changes in detector cells were confirmed considerably later after the actual assumed signalling event, i.e. after the stimulation of the detector cells by the inducer cells. In other words, the experimental approach used in our study minimized any possible artefacts due to the delayed investigation of physiological processes possibly linked to distant communication.

The results of the present study provide evidence of the existence of communication between neuroblastoma cells, physically separated in such a way as to inhibit any exchange of chemical signals through a solid or liquid medium. The occurence of NCDCI was more apparent after the stimulation of the inducer cells with dopamine, although the range of the resulting synchronization effect did not extend beyond the adjacent detector cell group (Position 2), i.e. a few millimetres.

Our purpose was to apply the novel biosensor set-up in order to investigate the possible existence of non-chemical, non-electrical communication between neural cells. We feel that the observed results support the assumption of the existence of distant communication between physically separated SK-N-SH human neuroblastoma cells. The elucidation of the mechanism behind this effect was beyond the scope of the present study.

That said, and taking into account the fact that the distant communi­cation was expressed as coordinated patterns of changes of the cell membrane potential, it can be assumed that the observed NCDCI in our experiments may be partly of electric or electromagnetic origin. It is worth mentioning that, using the novel biosensor system, we have recently shown that addition of 100 μΜ dopamine to murine N2a neuroblastoma cells caused cell membrane hyperpolarization, which in turn is responsible for the generation of potent localized electrical fields. Previous reports (e.g. Fröhlich and McCormick, 2010) have provided experimental support towards the hypothesis of a critical contribution of endogenous electric fields on coordinated responses of structured neuronal networks in vivo, while even an electromagnetic nature of consciousness has been proposed, as in the case of the con­scious electromagnetic information (CEMI) field theory (McFadden, 2013; Jones, 2017). On the other hand, the experimental configuration used in the study did not exclude the possibility of the transfer of volatile compounds from the inducer cells to the detector cells. The possibility of a gaseous distant chemical cell-to-cell interaction has been previously hypothesized based on the reported effect of CO2 on yeast cultures (Fels, 2016; Volodyaev, Krasilnikova and Ivanovsky, 2013). However, considering the results of the present study it is diffi­cult to suggest any volatile compound as being responsible for the observed NCDCI, given the very short range of the observed effect. In any case, our research group has recently started a series of experi­ments aiming to further elucidate the nature of the distant communica­tion between SK-N-SH neuroblastoma cells.

Acknowledgments

We wish to acknowledge the critical suggestions of Professor George Papadopoulos (Laboratory of Plant Breeding and Biometry, AUA) on the statistical process of the experimental data.

References

Apostolou, T., Moschopoulou, G., Kolotourou, E. & Kintzios, S. (2017) Assess­ment of in vitro dopamine-neuroblastoma cell interactions with a bioelectric bio­sensor: Perspective for a novel in vitro functional assay for dopamine agonist/ antagonist activity, Talanta, 170, pp. 69–73.

Chaban, V.V., Cho, T., Reid, C.B. & Norris, K.C. (2013) Physically disconnected non-diffusible cell-to-cell communication between neuroblastoma SH-SY5Y and DRG primary sensory neurons, American Journal of Translational Research, 5, pp. 69–79.

Cifra, M., Fields, J.Z. & Farhadi, A. (2011) Electromagnetic cellular interactions, Progress in Biophysics and Molecular Biology, 105, pp. 223–246.

Craddock, T.J., Friesen, D., Mane J., Hameroff, S. & Tuszynski, J.A. (2014) The feasibility of coherent energy transfer in microtubules, Journal of the Royal Society Interface, 11, 20140677.

Farhadi, A. (2014) Non-chemical distant cellular interactions as a potential con­founder of cell biology experiments, Frontiers in Physiology, 5, pp. 1–3.

Farhadi, A., Forsyth, C., Banan, A., Shaikh, M., Engen, P., Fields, J.Z. & Keshavarzian, A. (2007) Evidence of non-chemical, non-electrical intercellular signaling in intestinal epithelial cells, Bioelectrochemistry, 71, pp. 142–148.

Fels, D. (2016) Physical non-contact communication between microscopic aquatic species: Novel experimental evidences for an interspecies information exchange, Journal of Biophysics, Article ID 7406356.

Fels, D. (2009) Cellular communication through light, PLoS One, 4, e5086.

Ferentinos, K.P., Yialouris, C.P., Blouchos, P., Moschopoulou, G., Tsourou, V. & Kintzios, S. (2013) Pesticide residue screening using a novel artificial neural network combined with a bioelectric cellular biosensor, BioMed Research Inter­national, 813519.

Fröhlich, F. & McCormick, D.A. (2010) Endogenous electric fields may guide neocortical network, Neuron, 67, pp. 129–143.

Hashemibeni, B., Sadeghian, M., Aliakbari, M., Alinasab, Z., Akbari, M. & Salari, V. (2014) Experimental investigation of distant cellular interaction among adipose derived stem cells, Quantitative Biology, eprint arXiv: 1411.1498.

Jones, M.W. (2017) Mounting evidence that minds are neural EM fields inter­acting with brains, Journal of Consciousness Studies, 24 (1–2), pp. 159–183.

Kintzios, S., Pistola, E., Panagiotopoulos, P., Bomsel, M., Alexandropoulos, N., Bem, F., Biselis, I. & Levin, R. (2001) Bioelectric Recognition Assay (BERA), Biosensors and Bioelectronics, 16, pp. 325–336.

Kučera, O. & Cifra, M. (2013) Cell-to-cell signaling through light: Just a ghost of chance?, Cell Communication and Signaling, 11, pp. 87–95.

Kurian, P, Obisesan, T.O. & Craddock, T.J.A. (2017) Oxidative species-induced excitonic transport in tubulin aromatic networks: Potential implications for neurodegenerative disease, Journal of Photochemistry and Photobiology B, 175, pp. 109–124.

Matsuhashi, M., Pankrushina, A.N., Takeuchi, S., Ohshima, H., Miyoi, H., Endoh, K., Murayama, K., Watanabe, H., Endo, S., Tobi, M., Mano, Y., Hyodo, M., Kobayashi, T., Kaneko, T., Otani, S., Yoshimura, S., Harata, A. & Sawada, T. (1998) Production of sound waves by bacterial cells and the response of bacterial cells to sound, The Journal of General and Applied Microbiology, 44, pp. 49–55.

McFadden, J. (2013) The CEMI field theory: Closing the loop, Journal of Con­sciousness Studies, 20 (1–2), pp. 153–168.

Nikolaev, Y.A. (2000) Distant interactions in bacteria, Microbiology, 69, pp. 497–503.

Pospíšil, P., Prasad, A. & Rác, M. (2014) Role of reactive oxygen species in ultra-weak photon emission in biological systems, Journal of Photochemistry and Photobiology B: Biology, 139, pp. 11–23.

Prasad, A., Rossi, C., Lamponi, S., Pospíšil, P. & Foletti, A. (2014) New per­spective in cell communication: Potential role of ultra-weak photon emission, Journal of Photochemistry and Photobiology B: Biology, 139, pp. 47–53.

Reguera G. (2011) When microbial conversation gets physical, Trends in Micro­biology, 19, pp. 105–113.

Tang, R. & Dai, J. (2014) Biophoton signal transmission and processing in the brain, Journal of Photochemistry and Photobiology B: Biology, 139, pp. 71–75.

Trushin, M.V. (2003) The possible role of electromagnetic fields in bacterial communication, Journal of Microbiology, Immunology, and Infection, 36, pp. 153–160.

Trushin, M.V. (2004) Distant non-chemical communication in various biological systems, Rivista di Biologia, 97, pp. 409–442.

Volodyaev, I.V., Krasilnikova, E.N. & Ivanovsky, R.N. (2013) CO2 mediated interaction in yeast stimulates budding and growth on minimal media, PLoS One, 8, e62808.

Wainwright, M. (1998) Historical and recent evidence of the existence of mito­genetic radiation, Perspectives in Biology and Medicine, 41, pp. 565–571.

Paper received October 2017; revised January 2018.

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