Varen Eersapah

Sylain Hugel

Rémy Schlichter
email: schlichter@inci-cnrs.unistra.fr

High resolution detection of ATP release from single cultured mouse dorsal horn spinal cord glial cells and its modulation by noradrenaline

Keywords: astrocyte, exocytosis, secretory lysosome, vesicular release, non-vesicular release

High-resolution detection of ATP release from single cultured mouse dorsal horn spinal cord glial cells and its modulation by noradrenaline

Varen Eersapah, Sylain Hugel, Rémy Schlichter

Introduction

The dorsal horn of the spinal cord (DH) is an important structure for the integration and the transmission of peripheral nociceptive information {Basbaum, 2009 #29;Cordero-Erausquin, 2016 #101;Millan, 1999 #1}. It is also a strategic site where potentially painful messages can be modulated, in particular by descending controls originating from supraspinal centers {Fields, 2004 #34;Millan, 1999 #1;Pertovaara, 2006 Noradrenaline (NA) can inhibit glutamate release from primary afferents {Feng, 2002 #39;Funai, 2014 #8;Hickey, 2014 #37;Kawasaki, 2003 #11;Sonohata, 2004 Adrenoceptors are expressed by DH interneurons {Baba, 2000 #9;Kawasaki, 2003 #11;North, 1984 #19;Sonohata, 2004 #12} as well as by DH glial cells, at least in cultures of whole spinal cords {Hosli, 1982 #14}. ]. In the DH, NA fibres form synaptic contacts with fine dendrites of intrinsic DH neurons but not with primary afferent terminals (REFXX Satoh CellTissueres 1982, Hagashida Brain Res 1990, evt Holstege ProgBrainRes 1991). However, a large proportion of the contacts of noradrenergic fibres do not have the features of classical synapses suggesting the involvement of volume transmission (REFXX Rajaofetra JNeurocytol 1992).

Such "non-classical" contacts seem also common for serotonergic and dopaminergic fibres in the DH (REFXX Ridet Brain Res 1992, Ridet Neuroscience 1993). Most importanly, more that 60% of contacts formed by noradrenergic and serotoninergic fibres in the DH appear to be of the "non-synaptic" type and to be established with astrocytic profiles {Ridet, 1993 #16}. These morphological results suggest that DH glial cells might represent important targets of the descending noradrenergic projections and therefore might participate in the modulation of nociceptive messages by NA at the spinal level.

Recently, we have shown that NA selectively increased inhibitory synaptic transmission in the DH {Seibt, 2015 #41}. This phenomenon involved α1 and α2 adrenoceptors and functional communication between deep and superficial laminae of the DH {Seibt, 2015 #41}. Moreover, blocking the metabolism of DH glial cells with fluorocitrate suppressed the modulatory effect of NA on inhibitory synaptic transmission {Seibt, 2015 #41}, indicating a fundamental role of DH glia-to-neuron communication during noradrenergic modulation of inhibitory synaptic transmission. In addition, our data indicated that ATP seemed to play a major role as a signaling molecule in this interaction {Seibt, 2015 #41}.

Therefore, the aim of the present work was to determine whether NA was able to trigger/modulate ATP release from DH glial cells and to characterize the mechanisms involved in this phenomenon. To this end, we developed and improved a sniffer cell/biosensor system that allowed us to detect low nanomolar concentrations of extracellular ATP and to quantify ATP release at the single cell level with excellent spatial and temporal resolutions.

Methods

Animal procedures

Mice were bred and housed in the animal facility Chronobiotron, UMS3415, accredited according to EU Directive 2010/63 and French regulations 2013-118. All procedures were conducted in conformity with the rules of the European Communities Council Directive 2010/63/EU, and the French regulations 2013-118. Pups were kept with their mothers until euthanasia by decapitation which is considered as an ethical method for very young animals.

Primary cultures of DH glial cell

Cultures of primary DH spinal cord glial cells were prepared from neonatal 3 to 6 day old postnatal C57BL/6 mice pups of either sex. Animals were decapitated and a laminectomy was performed in order to expose the dorsal part of the lumbar spinal cord. After having removed the meninges with thin forceps, the dorsal half of the spinal cord (i.e. the region of the dorsal horn) was harvested and placed in cold (4°C) Dulbecco's Modified Eagle Medium / F-12 containing 15 mM HEPES (Gibco, France).

The collected spinal tissue was then dissociated mechanically using fire-polished Pasteur glass pipettes of decreasing tip diameters until obtaining a turbid solution containing the dissociated cells and tissue debris. This solution was centrifuged at 500 RPM for 5 min. The supernatant containing large debris was removed and discarded and the pellet containing the glial cells was re-suspended in 2 mL of culture medium composed of: Dulbecco's Modified Eagle Medium supplemented with 10 % (v/v) heat inactivated horse serum (Gibco, France) and 0.5 % (v/v) streptomycin/penicillin (at 5.000 µg/ml and 5,000 U/mL respectively, Gibco, France).

The cell suspension was seeded in the center of sterile 35 mm standard plastic culture dishes (Corning, USA) in which a central hole (18 mm) had been drilled and to the bottom of which a glass coverslip had been glued, thus delimiting a central well in the culture dish. The internal part of the glass bottom of the dishes was coated with poly-D-lysine (0.02 mg/mL, Sigma, France) in order to favor the attachment of the cells. After seeding, the cultures were placed and maintained in a 37 o C water-saturated incubator in an atmosphere containing air and 5 % CO2.

Experiments were performed on confluent cells 2-3 weeks after seeding.

Maintenance of Human Embryonic Kidney 293 (HEK293) cells

In order to detect ATP release from cultured DH glial cells we used a human embryonic kidney (HEK293) cell line that was stably transfected with the rat P2X2 receptor subunit {Stoop, 1999 #24}. This cell line, which we will refer to as HEK293-P2X2 later in the text, was kindly provided by Dr. François Rassendren (Institut de Génomique Fonctionnelle, Montpellier France). These cells express homomeric P2X2 receptors {Stoop, 1999 #24} and were previously used by us to characterize P2X2 receptor-mediated currents and their modulation by progesterone {De Roo, 2010 #102}. HEK293-P2X2 cells were cultured in sterile 75 mm 2 flasks (Thermo Scientific, Denmark) in a culture medium consisting of: Dubelcco's modified Eagle medium with 25 mM HEPES (Gibco) to which were added foetal calf serum (10% v/v, Gibco), penicillin-streptomycin (50 IU/mL each, Gibco) and glutamax (1% v/v, Gibco). The transfected sequence contained the rat P2X2 subunit sequence as well as a neomycin resistance cassette {Stoop, 1999 #24}. This allowed for the selection of P2X2-expressing HEK293 cells by culturing them in the presence of geneticin (0.5 mg/mL, G418, Gibco). The cultures were kept at 37°C in a water saturated incubator in an atmosphere composed of air and 5% CO2. Before calcium imaging experiments HEK293P2X2 cells were mechanically detached from the bottom of the culture flask by flushing the cells with a stream of culture medium by means of a 10 ml sterile plastic pipette. The medium containing the detached cells was then transferred to a 15 mL sterile plastic tube (Greiner Bio-One, Germany) and the volume was adjusted to 10 mL with standard culture medium. This solution was centrifuged at 500 RPM for 5 min and the pellet was re-suspended in culture medium or extracellular medium used for calcium imaging experiments (see below). In order to prepare cultures of HEK293-P2X2 cells alone, cells were re-suspended in 2 mL standard culture medium, seeded on poly-D-lysine coated glass-bottom culture dishes and maintained as described for DH glial cultures in the preceding section. These cultures were used to determine the intrinsic properties of HEK293-P2X2 cells in calcium imaging experiments. Alternatively, HEK293-P2X2 cells were used as biosensor cells to detect ATP release from cultured DH glial cells. To this end, the dissociated HEK293-P2X2 cells were re-suspended in 2 mL of the serum-free HEPES buffer-based extracellular solution used for calcium experiments, loaded with fura-2 and plated on top of cultured DH glial cells just before the beginning of the experiments (see below).

Calcium imaging

I. Loading of cells with fura-2 Cultured DH glial cells or HEK293-P2X2 cells were washed several times with the extracellular HEPES-based extracellular solution used in all the calcium imaging experiments. This extracellular solution was composed of (in mM): NaCl, 135; KCl, 5; CaCl2, 2.5; MgCl2, 1; glucose, 10 and HEPES, 5. Loading of cells was achieved by incubating them for 1 h at room temperature (22-25°C) in extracellular medium containing 4 µM fura-2 acetoxymethyl ester (fura-2 AM) (Molecular Probes, USA), 0.001% (w/v) pluronic acid (Molecular Probes, USA) in order to facilitate fura-2 AM uptake by the cells. After loading, the cells were washed with extracellular medium and used for calcium experiments on the experimental set up.

In experiments in which HEK293-P2X2 cells were used as biosensors for ATP release from DH glial cells, the loading with fura-2 was performed on suspensions of HEK293-P2X2 cell (see above).This protocol was performed at room temperature (22-25°C) following a protocol similar to that described above for glial cultures, except that the "loading-medium" also contained 200 nM progesterone (PROG, Sigma Alkadrich, USA) in order to potentiate P2X2 receptor function {De Roo, 2010 #102}. After 1 h incubation, this suspension of HEK293-P2X2 cells was diluted with 9 mL of extracellular solution and centrifuged at 500 RPM for 5 min. The pellet was rinsed two times with 10 mL of extracellular solution and re-suspended in 2 mL of extracellular solution. This suspension of fura-2-and progesterone-loaded HEK293-P2X2 cells was then seeded on top of cultured DH glial cells (200 µL per dish).

II. Measurements of changes in intracellular free calcium concentration ([Ca 2+ ]i) To

monitor changes in [Ca 2+ ]i, culture dishes were transferred to the stage of an inverted fluorescence microscope (Axiovert 35; Zeiss, Gottingen, Germany) and the cells were visualized with a x40 oil-immersion objective (Fluor 40; NA, 1.30; Nikon, Tokyo, Japan). The cultures were continuously superfused with extracellular medium, the temperature of which was maintained at 34°C. Cells were allowed to habituate to this temperature for at least 15 min before starting the experiments. In experiments using HEK293-P2X2 cells as ATP biosensors, the fura-2 and progesterone loaded cells (HEK293-P2X2-PROG cells) were seeded on top of the glial cells once the culture dishes of glial cells had been placed on the microscope stage.

This procedure was performed at room temperature and in the absence of extracellular superfusion. Fifteen min after seeding, HEK293-P2X2-PROG cells had attached sufficiently to the glial cell layer and superfusion with heated extracellular solution was started. It should be noted that in biosensor experiments, only HEK293-P2X2-PROG cells were loaded with fura-2. Thus, the measured changes in [Ca 2+ ]i were solely due to changes in calcium levels in HEK293-P2X2-PROG cells since glial cells did not contain fura-2.

Calcium signals were acquired with a quantitative real-time imaging system comprising a cooled CCD camera (CoolSNAP HQ; Roper Scientific, Tucson, AZ, USA) and an image analysis software package (Imaging workbench 4.0; Axon Instruments, Molecular Devices).

Cells were alternately excited at wavelengths of 350 and 380 nm with a lambda-10 filter wheel (Sutter instruments, USA), and emitted light was collected above 520 nm. Pairs of images were acquired every 1.1 seconds. Throughout the manuscript, intracellular calcium levels and their variations are expressed as the ratio of fluorescence signals (ratio F350/F380) measured at 520 nm after alternate excitation at 350 and 380 nm. This ratio was calculated after background signal subtraction. All experiments were performed at 34°C.

III. Calcium imaging analysis

For analysis, regions of interest (ROIs) corresponding to individual cells were selected manually offline using the Imaging workbench 4.0 software (Axon Instruments, Molecular Devices). ROIs were chosen based on cell morphology and cell responsiveness to a positive control in order to avoid any selection bias by the experimenter.

For cultured glial cells, this positive control consisted in the existence of a large transient increase in [Ca 2+ ]i following the local application of 30 µM ATP at the end of the recording session. For HEK293-P2X2-PROG cells, only those that responded with an increase in [Ca 2+ ]i to a low (200 nM) concentration of exogenously applied ATP were considered in the analysis. This response corresponded to the change in [Ca 2+ ]i triggered by the selective activation of P2X2 receptors and attested cells ability to sense low concentrations of extracellular ATP (for details see Results section and Fig. 2). In a given field, all cells that displaying an increase in [Ca 2+ ]i following the application of ATP were selected for analysis regardless of the fact that they responded or not to other substances applied during the experiment.

The acquired florescence signals were analyzed in more detail with the Clampfit 10.7.0 software (Molecular devices) which allowed us to quantify baseline F350/F380 ratio values as well as their changes during application of pharmacological substances. Measurements of area under the curve (AUC) of the calcium signals in fura-2 loaded HEK293-P2X2-PROG cells in contact with glial cells were used to quantify ATP release from glia under control and different experimental conditions (e.g. after treatment with vesicular release inhibitors). To this end, the experimental fluorescence values were divided by the mean F350/F380 ratio value measured before application of the tested substance. This value was determined over a period of 500 s preceding substance application and this procedure therefore normalized the basal fluorescence ratio value to 1 and the area under the curve (AUC) value corresponding to this control period to 500 (ratio of 1 during 500 s). Changes in ATP release were quantified by the changes in the AUC value in HEK293-P2X2-PROG cells that were induced by local application of noradrenaline, different adrenoceptor agonists or a low K + containing extracellular solution (see below). These applications lasted 150 s and the AUC was measured over a 500 s period, which included the 150 s lasting application of the substance and a subsequent 350 s period outlasting the application time in order to include delayed/prolonged effects on ATP release from glial cells. The AUC determined during the 500 s period before application was compared to that measured during the 500 s following the onset of application of the substance to be tested. The increase in AUC triggered by the substance reflected the increase in ATP release from glia,

Pharmacological substances

Pharmacological substances to be tested were prepared as 1000 times concentrated stock solutions and diluted to their final concentration in extracellular solution just before the beginning of the experiments. D,L -Noradrenaline hydrochloride, phenylephrine hydrochloride, clonidine hydrochloride, isoproterenol hydrochloride, adenosine triphosphate disodium salt (ATP), propranolol hydrochloride, prazosin hydrochloride, yohimbine hydrochloride (all Sigma-Aldrich, USA) and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (Tocris, UK) were prepared in distilled water and stored at -20°C.

Fura-2-AM (Molecular Probes, USA), 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (BAPTA-AM) (Sigma-Aldrich, USA), bafilomycin A1 (Abcam, Cambridge, UK), glycyl-L-phenylalanine 2-naphthylamide (GPN) (Abcam, Cambridge, UK) were prepared in dimethyl sulfoxide (DMSO) and stored at 4°C. Progesterone (PROG, Sigma Aldrich, USA) was prepared in ethanol 70% (v/v) and stored at 4°C.

Application of substances

During all calcium imaging experiments the cells were continuously superfused with extracellular solution preheated at 34°C. The total bath volume was 2 mL and perfusion rate was 3 mL.min -1 . Substances to be tested were applied locally by means of a gravity driven multichannel perfusion device consisting of independent 5 mL syringes connected via separated polyethylene tubings to a common final outlet, i.e. a 0.9 mm metal needle placed just above the cells. The substance to be applied was selected by opening a tap placed between the syringe and the polyethylene tube connected to this syringe. Outside application periods, standard extracellular solution was continuously flowing through the application system to the cells via one of the application channels that remained open. During application of a substance, the tap of the control extracellular solution channel was closed simultaneously with the opening of the tap of the substance to be tested. The reverse procedure was followed at the end of the application period of the substance.

In all experiments we checked that switching between two channels containing extracellular solution did not produce mechanical artifacts that could trigger an increase in [Ca 2+ ]i. We also verified that application of vehicle/solvents such as ethanol, H2O, or DMSO in which stock solutions of substances were prepared, did not induce changes [Ca 2+ ]i. In some experiments increases in [Ca 2+ ]i were triggered by applying a low K + solution {Dallwig, 2000 #98;Hartel, 2007 #63}. To this end the concentration of KCl in the extracellular solution was reduced from 5 mM to 0.2 mM and the NaCl concentration was increased from 135.0 mM to 139.8 mM.

Application of this low

Fitting of dose-response curves

Dose-response curves for ATP were described with the following equation: y=100/(1+(EC50/[ATP])^AH) where y is the proportion of responding cells and AH a Hill coefficient.

Statistical analysis

Statistical data analyses were performed using the statistical software Kyplot 5.0. (KyensLab, Tokyo, Japan). Proportions in contingency tables were compared with Fisher's exact test (http://quantpsy.org/fisher/fisher.htm). Comparison between means was performed with paired or unpaired (as appropriate) Student's t-test. The significance level for statistical tests was set as α = 0.05. Symbols to indicate P-values that are used in the figures are as follows:

****P < 0.00001 < ***P < 0.0001 < **P <0.001 < *P < 0.05 < n.s (not significant).

Most results are expressed as proportion of responsive cells given as X/n where X is the number of responsive cells and "n" the total number of cells tested.

The other numerical results are given as mean ± s.e.m. (standard error to the mean).

Results

DH glial cell cultures

Our aim was to use a mixed DH glial cell culture rather than isolated "purified" glial cell populations in order to maintain the coexistence and the interaction between the major DH glial cell types in culture. Different considerations indicate that our cultures contained no neurons and mainly astrocytes. The absence of neurons was suggested by calcium imaging experiments showing that the application of a depolarizing extracellular solution containing a high (50 mM) K + concentration did not induce an increase in [Ca 2+ ]i that is typical for neurons in culture, Indeed, we have a long standing experience in the preparation of primary postnatal DH mixed neuron-glia cultures {Hugel, 2000 #21;Jo, 1999 #22} and we have clearly established that, in order to obtain viable neurons, it is necessary to use an enzymatic dissociation followed by a very mild mechanical dissociation. When mechanical dissociation is used alone or is too intense, neurons do not survive.

In addition, it has been clearly established that, among glial cells, α1 adrenoceptors are expressed by astrocytes but not by microglia even in cell culture {Hertz, 2010 #72}. In our cultures, 90% of the cells responded to the α1 adrenoceptor specific agonist phenylephrine (PHE, see below) suggesting that astrocytes were the main glial cell type present in these cultures. Moreover, during calcium imaging experiments, we observed that the cells displaying an increase in [Ca 2+ ]i upon stimulation with PHE had a flattened star-shaped morphology that is characteristic of astrocytes in culture.

Taken together, these arguments indicate that the majority of cells present in our cultures appeared to be astrocytes.

Effect of noradrenaline (NA) and adrenoceptor agonists on intracellular free calcium concentration ([Ca 2+ ]i) in DH glial cells

Local application of NA (20 µM, 50 s) induced an increase in [Ca 2+ ]i in 86.0% of the cells tested (172/200). Three types of responses were observed (Fig. 1a). In the majority of cases, cells responded with a type 1 response that consisted of an initial fast transient peak followed by lower amplitude plateau phase that slowly declined but persisted during the whole duration of NA application. Type 1 responses were observed in 85.5% (147/172) of the NA-responsive DH glial cells. A second type of response (type 2) consisted of an initial peak followed by oscillations in [Ca 2+ ]i that occurred during the plateau phase. Such oscillatory responses were recorded in 11.0% (19/172) of the cells. Finally, a small proportion of cells, i.e. 3.5% (6/172), displayed a type 3 response that consisted of a single initial peak with no detectable plateau phase or oscillations in [Ca 2+ ]i.

We next tested the classical α1, β and α2 adrenoceptor agonists phenylephrine (PHE, 20 µM), isoproterenol (ISO, 10 µM) and clonidine (CLO, 20 µM), respectively. As illustrated in Fig. 1b, PHE, ISO and CLO induced increases in [Ca 2+ ]i in 95.0% (132/139), 23.7% (47/198) and 6.1% (7/114) of the cells, respectively. The proportion of responses to PHE was significantly reduced to 5.1% (4/79) in the presence of the α1 adrenoceptor antagonist prazosin (2 µM) (Fisher's exact test, P = 9.4 10 -45 ). Similarly, the proportion of responses to ISO was significantly reduced to 7.0% (4/57) by the β adrenoceptor antagonist propranolol (20 µM) (Fisher's exact test, P = 0.03). By contrast, the proportion of responses to CLO was very low (6.1% see above) and not significantly modified by the α2 adrenoceptor antagonist yohimbine (1 µM) (1.7%, 1/58, Fisher's exact test; P = 0.265). In agreement with these observations, we found that a cocktail of prazosin, propranolol and yohimbine virtually abolished the responses to NA (5.1%, 4/79, Fisher's exact test; P = 4.0 10 -38 ). Taken together, our results suggest that NA increased [Ca 2+ ]i in about 90% cultured DH glial cells and that this effect was largely mimicked both qualitatively and quantitatively by the α1agonist PHE. In addition, about 25% of the cells displayed a response to ISO but virtually none to CLO. We therefore decided to investigate in more detail the effects of PHE and NA on the release of ATP from DH glial cells.

Detection of extracellular ATP by an improved biosensor system

In order to detect the release of ATP from DH glial cells and its modulation by NA and PHE, we used a biosensor/sniffer cell approach. We chose this option because, once released in the extracellular space, ATP is rapidly metabolized by ectonucleotidases {Dunwiddie, 1997 #42;Zimmermann, 1999 #43;Zimmermann, 2000 #45} making it difficult to detect accurately this short-lived molecule. We decided to use HEK293 cells stably transfected with rat P2X2 receptors (HEK293-P2X2) {Stoop, 1999 #24} as a detection system for extracellular ATP, since these receptors are highly permeable to Ca 2+ ions {Egan, 2004 #28} and Ca 2+ influx through these receptors can be detected by calcium indicators such as fura-2. The general idea of our approach was to plate fura-2-loaded HEK293-P2X2 cells on top of cultured DH glial cells. Under these conditions changes in [Ca 2+ ]i measured in HEK293-P2X2 cells will reflect and allow to monitor the changes in ATP release from glial cells following the application of NA and PHE (see below) with good spatial and temporal resolutions.

When ATP was applied to HEK293-P2X2 cells cultured alone and loaded with fura-2, we observed an increase in [Ca 2+ ]i. The dose-response relationship illustrating the proportion cells displaying an increase in [Ca 2+ ]i for increasing concentrations of ATP applied is illustrated in Fig. 2a. The apparent EC50 of ATP was 0.75 ± 0.02 µM. Under these conditions, it was difficult to detect low nanomolar concentrations of ATP. Indeed, application of 200 nM ATP triggered small calcium responses in only 5.3% (4/76) of the cells (Fig. 2b), corresponding approximately to the EC5 of our dose response curve. However, we have previously shown that rat P2X2 receptors can be strongly and selectively potentiated by the steroid/neurosteroid progesterone (PROG) {De Roo, 2010 #102}. Fig. 2b shows that acute application of PROG (5 µM) to HEK293-P2X2 cells for 100 s did not evoke any significant increase in [Ca 2+ ]i. However, when ATP (200 nM), that initially triggered small and rare responses before PROG application, was applied to HEK293-P2X2 cells after they had been exposed to PROG (5 µM), it evoked large increases in [Ca 2+ ]i in the large majority of the cells ( 75 Taken together these results suggest that PROG-potentiated P2X2 receptors stably expressed by HEK293 cells represent an excellent tool to detect low nanomolar concentrations of extracellular ATP.

Quantification of ATP release from cultured DH glial cells

In order to detect and quantify ATP release from cultured glial cells, HEK293-P2X2 cells were loaded with fura-2-AM, and incubated with 200 nM PROG for one hour at room temperature and then plated on top of confluent DH glial cell cultures (see Methods section). Since only HEK293-P2X2 cells were loaded with fura-2, changes in [Ca 2+ ]i reflected either spontaneously occurring calcium fluctuations in these cells or/and activation of P2X2 receptors by ATP released from DH glial cells in close contact with the HEK293-P2X2 cells.

In an initial set of experiments, we quantified the spontaneously occurring changes in [Ca 2+ ]i in HEK293-P2X2 cells pre-incubated with PROG but cultured alone (i.e. in the absence of DH glial cells). Under these conditions, the [Ca 2+ ]i levels were usually very stable and rarely displayed spontaneously occurring peaks in [Ca 2+ ]i (6.8%; 10/146). When observed, the frequency of peaks was very low (2.8 10 -3 ± 4.4 10 -4 Hz, n= 10). Moreover, these cells did rarely displayed increases in [Ca 2+ ]i following the application of NA (20 µM) nor PHE (20 µM). Indeed, we observed an increase in F350/F380 ratio in only 2% (2/99) of the cells, and this change consisted of a single peak (type 3 response in Fig. 1a). Calcium peaks occurring spontaneously in HEK293-P2X2-PROG cells plated on top of DH glial cells were also relatively rare and represented 11.9% of the cases (19/160). This proportion of cells was not significantly different from that of HEK293-P2X2-PROG displaying calcium peak in the absence of DH glial cells (Fischer's exact test; P = 0.17). Moreover, the mean frequency of peaks was not different from that in the absence of glia (3.0 10 -3 ± 2.1 10 -4 Hz, n= 42; Fisher's exact test; P = 0.75).

In contrast, when PROG loaded HEK293-P2X2 cells (HEK293-P2X2-PROG) were seeded on top of DH glial cell cultures, application of NA (20 µM) or PHE (20 µM) induced the appearance of peak-like increases of [Ca 2+ ]i in the HEK293-P2X2-PROG cells (Figs 3a1,b1).

In 23 cases, HEK293-P2X2-PROG cells displayed spontaneous calcium peaks before the application of NA (20 µM). The mean frequency of peaks was of 3.0 10 -3 ± 2.8 10 -4 Hz (n=23).

After application of NA (20 µM), the frequency of peaks significantly increased to 4.3 10 -3 ± 3.3 10 -4 Hz (n= 23, Fisher's exact test; P = 0.007). Similarly, in 19 cases, HEK293-P2X2-PROG cells displayed spontaneous calcium peaks before the application of PHE (20 µM). The mean frequency of peaks was of 3.0 10 -3 ± 3.2 10 -4 Hz (n=19). After application of PHE (20 µM), the frequency of peaks significantly increased to 4.4 10 -3 ± 5.6 10 -4 Hz (n= 19, Fisher's exact test; P = 4.0 10 -3 ).

In order to facilitate the representation of the effects of NA and PHE on calcium peaks, data from all responsive cells were pooled. These data are presented in Fig. 3a2 and Fig. 3b2 under the form of histograms of the number of calcium peaks as a function of time before, during and after the application of NA and PHE. Application of NA (20 µM) or PHE (20 µM) induced an increase in the number of [Ca 2+ ]i peaks that outlasted the duration of NA or PHE application.

Interestingly, an increase in the number/frequency of calcium peaks by NA or PHE could be induced only by the first application of these agonists. A second application, even if performed 1000 s after the first application did not induce the appearance or the increase in frequency of [Ca 2+ ]i peaks, whereas effects on calcium responses in DH glial cells alone could be reproduced at least two times (see above). The calcium signals induced by PHE were never observed when DH glial cells were pre-loaded with BAPTA-AM (0/87, Fig. 3c) or when PPADS (50 µM) was added to the extracellular medium (0/71, Fig. 3d).

In order to quantify more precisely the changes in [Ca 2+ ]i that occurred in HEK293-P2X2-PROG cells, we determined the area under the curve (AUC) of the fura-2 signal and normalized each signal by dividing it by the value of the baseline, thus allowing to compare the results between different cells (see Methods section for more details). This was done for equivalent periods of time (i.e. 500 s) before and after application of PHE. As shown in Fig. 3e, PHE significantly increased the AUC from 504.8 ± 1.1 a.u. (arbitrary units) to 518.2 ± 5.0 a.u. (n=68, paired Student's t-test; P = 6.2 10 -4 ). The effect of PHE was abolished by PPADS (50 µM) or after loading the DH glial cells with the calcium chelator BAPTA-AM (Fig. 3e). In the presence PPADS (50µM), the AUC was of 503.8 ± 1.6 a.u. before and of 502.0 ± 2.3 a.u. after application of PHE (20 µM) (n= 70). These values did not differ significantly (paired Student's t-test; P = 0.37). When DH glial cells were preincubated with BAPTA-AM, the AUC was of 505.0 ± 1.1 a.u. before and of 506.0 ± 0.9 a.u. after application of PHE (20 µM) (n= 87). These values did not differ significantly (paired Student's t-test; P = 0.27).

These results show that the increase in [Ca 2+ ]i involved the activation of PPADS-sensitive P2X2 receptors expressed by HEK293-P2X2 cells. Moreover, BAPTA specifically loaded into DH glial cells blocked the changes in [Ca 2+ ]i observed in HEK293-P2X2 cells indicating that these changes were due to a calcium-dependent release of ATP from the cultured DH glial cells.

Effect of reducing extracellular K + concentration on the release of ATP

Since NA and PHE triggered calcium-dependent release of ATP from cultured DH glial cells (see preceding section), we wondered whether other stimuli that increase [Ca 2+ ]i were able to induce ATP release from these cells.

It has been reported that application of an extracellular solution containing a low concentration of K + ions (low K + ) can induce the a rise in [Ca 2+ ]i in astrocytes by activating Ba 2+ -sensitive Kir4.1 inwardly rectyfing K + channels {Dallwig, 2000 #98;Hartel, 2007 #63}. We therefore first tested the effect of applying a low K + solution (in which the concentration of K + was reduced from 5 mM to 0.2 mM) to cultured DH glial cells loaded with fura-2. Application of this low K + solution rapidly induced a rise in [Ca 2+ ]i that lasted for the whole duration of the application (Fig. 4a). Increases in [Ca 2+ ]i were observed in a large majority cultured DH glial cells tested (86.9%, 54/62). Moreover, this increase in [Ca 2+ ]i could be reproduced several times in the same cells when applied at an interval of 1000 s (Fig. 4a). This low K + -induced response was totally blocked (0/31) when 2 mM Ba 2+ was added to the extracellular solution.

Interestingly, when tested under the same experimental conditions, the low K + solution never induced a rise in [Ca 2+ ]i in PROG treated HEK293-P2X2 cells cultured alone (0/150).

When we applied a low K + medium to fura-2-loaded DH glial cells, we observed an increase in [Ca 2+ ]i in 87.1% (54/62) of them (Fig 4b). This proportion was unaffected in the presence of PPADS (50 µM) in the extracellular medium (88.6%, 39/4, P = 1.0) but significantly reduced after preloading of the glial cells with BAPTA-AM (3.2%, 1/31, P = 6.0 10 -16 ) Application of the low K + solution to co-cultures of DH glial cells and PROG treated fura-2loaded HEK293-P2X2, resulted in an increase in [Ca 2+ ]i the HEK293-P2X2 cells (Fig. 4c).

Such an effect was observed in 64.1% (84/131) of the recorded cells. None of the of HEK293-P2X2-PROG on top DH glial cells displayed an increase in [Ca 2+ ]i in the presence of PPADS (50 µM) in the extracellular medium (0.0%, 0/42, Fisher's exact test, = 1.5 10 -15 ) and the proportion of HEK293-P2X2-PROG with a calcium response was significantly reduced when the DH glial cells were preloaded with BAPTA-AM (0.0%, 0/37, Fisher's exact test; P = 8.6 10 -14 ) (data not illustrated).

We further quantified this response by determining the relative AUC for each response (Fig. 4d). In the absence of any treatment, the AUC of the calcium response induced by low K + was of 523.0 ± 4.0 a.u. (n= 131). The AUC was of 503.7 ± 0.6 a.u. (= 42) in the presence of PPADS and of 503.2 ± 1.1 a.u. (= 37) after preloading the DH glial cells with BAPTA-AM (Fig. 4d).

These values were significantly different from the AUC of the low K + calcium response measured in the absence of any treatment: PPADS, unpaired student's t-test; P = 4.9 10 -4 ; and BAPTA-AM, unpaired student's t-test; P = 1.9 10 -4 ).

Taken together, these results suggested that application of a low K + solution activated Ba 2+sensitive (most probably Kir4.1) channels that triggered an increase in [Ca 2+ ]i which in turn led to a calcium-dependent release of ATP that was sensed by the transfected P2X2 receptors.

Distinct vesicular pools of ATP are mobilized by NA and low K + stimulation of cultured

DH glial cells

In order to check whether calcium-dependent ATP release involved intracellular vesicles, we tested the effects of bafilomycin A1 and glycyl-L-phenylalanine 2-naphtylamide (GPN).

Bafilomycin A1 blocks the activity of the proton pump present on acidic vesicles that generates the pH gradient across the vesicle membrane that is necessary for the loading of the vesicles with the transmitter. Bafilomycin A1 acts on all types of acidic vesicles including light clear Fig. 5a summarizes the effects of GPN (Fig. 5a2) and bafilomycin A1 (Fig. 5a3) on changes in [Ca 2+ ]i measured in fura-2 loaded, PROG treated HEK293-P2X2 cells plated on top of cultured DH glial cells. When glial cells were pretreated with GPN, the proportion of responses to PHE was strongly decreased (6.3%, 5/80, Fisher's exact test; P = 2.8 10 -18 ) and that responding to low K + was slightly reduced (41.3%, 33/80, Fisher's exact test; P = 2.0 10 -3 ). After preincubation of the glial cells with bafilomycin A1, there was a significant reduction of the proportion of the responses to PHE (11.8%, 8/68, Fisher's exact test; P = 3.7 10 -13 ) as well as to low K + (5.9%, 3/51, Fisher's exact test; P = 7.7 10 -12 ).

When glial cells were pretreated with GPN, the baseline value (BL) of the AUC was of 503.4 ± 1.1 a.u. (n= 80) and that after PHE (20 µM) application was of 503.8 ± 1.7 a.u. (n=80). These values were not significantly different (paired Student's t-test; P = 0.84). In the experiments with low K + applied to GPN-treated glial cells, the AUC measured in HEK203-P2X2-PROG cells on top of the glia under resting conditions was of 502.7 ± 1.7 a.u. (n= 80). This value significantly increased to 514.2 ± 3.1 a.u. (n= 80, Paired Student's t-test; P = 3.1 10 -6 ) after application of low K + .

When glial cells were pretreated with bafilomycin A1, the baseline value (BL) of the AUC was of 504.0 ± 1.0 a.u. (n= 68) and that after PHE (20 µM) application was of 505.0 ± 1.3 a.u. (n=68). These values were not significantly different (paired Student's t-test; P = 0.16). In the experiments with low K + applied to bafilomycin A1-treated glial cells, the AUC measured in HEK203-P2X2-PROG cells on top of the glia under resting conditions was of 501.9 ± 1.1 a.u.

(n= 51) and of 501.2 ± 1.2 a.u. (n= 51, Paired Student's t-test; P = 3.1 10 -6 ) after application of low K + . These values were not significantly different (paired Student's t-test, P = 0.39).

To summarize, pretreatment of cultured glial cells with GPN suppressed the increase in [Ca 2+ ]i induced by PHE (20 µM) application but not that induced by the low K + solution, whereas pretreatment with bafilomycin A1 suppressed both responses to PHE and low K + . These results indicated that ATP can be released from a secretory lysosomal (GPN-sensitive) pool as well as from and a non-lysosomal (bafilomycin A1-senstitive) pool. Our data also show that PHE targets selectively the secretory lysosomal pool.

Spontaneous release of ATP from cultured DH glial cells

As mentioned above, HEK293-P2X2-PROG cells plated on DH glial cells rarely displayed spontaneously occurring peaks/changes in [Ca 2+ ]i, i.e. in the absence of an application of NA or PHE, indicating that spontaneous vesicular release was a rare phenomenon under our basal experimental conditions. One reason for apparently not detecting spontaneous release might be that ATP released from DH glia under basal conditions did not reach a local concentration sufficient to significantly activate P2X2 receptors in the detector cells. We therefore decided to favor accumulation of ATP released from DH glial cells by temporarily interrupting (stopping) the constant superfusion of the cells with extracellular solution. Such stop/flow experiments have been used previously to demonstrate ATP release from cultured astrocytes following mechanical or electrical stimulation of the glial cells {Hassinger, 1996 #71}. Fig. 6a,b shows that stopping the general and local superfusion induced a significant increase in the F350/F380 fluorescence ratio in 55.4% (46/83) of the detector HEK293-P2X2-PROG cells. When extracellular superfusion was switched on again, the fluorescence ratio returned to control levels, i.e. to a value close to that recorded before stopping the flow of extracellular solution, thereby indicating that the increase in [Ca 2+ ]i in the detector cells was reversible. This increase in [Ca 2+ ]i was due to the activation of P2X2 receptors by ATP because the fraction of cells displaying an increase in fluorescence ratio was significantly reduced (7.6%; 4/60, Fisher's exact test P = 3.7 10 -10 ) in the steady state presence of PPADS (50 µM) in the extracellular solution (Fig. 6a,b). There was also a significant reduction of the fraction of cells displaying an increase in F350/F380 ratio when glial cells were preloaded with the calcium chelator BAPTA-AM (4.5%, 2/44, Fisher's exact test P = 2.3 10 -9 ; Fig. 6). In contrast, pretreating the DH glial cells with bafilomycin A1 in order to block vesicular release of ATP (see above), did not significantly change the fraction of detector cells displaying an increase in [Ca 2+ ]i following the interruption of extracellular superfusion (Fig. 6a) (67.7%; 25/37, Fisher's exact test P = 0.23; Fig. 6).

Taken together our results suggest the existence of a spontaneous ATP release from DH glial cells. This release is calcium-dependent but does not involve acidic vesicles.

Discussion

Our results demonstrate that NA induced/stimulated the release of ATP from cultured DH glial cells. This effect relied on the activation of α1 adrenoceptors and the calcium-dependent fusion of secretory lysosomes with the plasma membrane. The detection of the release of low nanomolar concentrations of ATP at the single cell level was possible owing to the development of an improved sniffer cell system consisting of HEK293 cells expressing rat P2X2 receptors that are strongly and selectively potentiated by the steroid progesterone (PROG). This system provided very good sensitivity to ATP and excellent spatial and time-resolutions. It also allowed us to demonstrate at the single cell level the coexistence of distinct vesicular and non-vesicular calcium-dependent release mechanisms of ATP {Blum, 2008 #103;Dubyak, 2012 #96}.

Improved sniffer cell detection system for the detection of ATP release from DH glial cells

ATP is an important extracellular signaling molecule in the nervous system that can be released from various cell types including neurons and glial cells {Burnstock, 2006 #46;Fields, 2006 #48;Guthrie, 1999 #74;Hassinger, 1996 #71;Verkhratsky, 2018 #73}. ATP is also recognized as a major gliotransmitter that underlies calcium wave propagation in glial networks and modulation of electrical activity in neuronal networks {Boue- Grabot, 2017 #49;Guthrie, 1999 #74;Hassinger, 1996 #71;Haydon, 2001 #20;Lalo, 2014 #51;Verkhratsky, 2018 #73} including in the DH of the spinal cord {Hugel, 2000 #21;Jo, 1999 #22;Scemes, 2000 #60}. A major limit for the precise detection and quantification of ATP release is its rapid degradation by extracellular ectonucleotidases {Dunwiddie, 1997 #42;Zimmermann, 1999 #43;Zimmermann, 2000 #45} which strongly reduce the lifetime of ATP in the extracellular space once it has been released. This is a serious challenge when trying to quantify low concentrations of extracellular ATP, in particular at the single cell level. Several approaches have been developed to try to resolve this issue. One of these consists of using calcium-permeable ionotropic ATP receptors (P2X receptors) as detection/detector elements. HEK293 cells transfected with P2X2 or mutant P2X3 receptors have been used successfully for detecting ATP release from glial cells {Lalo, 2014 #51;Pangrsic, 2007 #76}. We found, when using a calcium imaging approach, that simply expressing P2X2 receptors in HEK293 cells did not improve the ATP detecting capacity of these cells that naturally express metabotropic P2Y receptors {Atwood, 2011 #77;Fischer, 2005 #78}. Indeed, our results show that non-transfected and HEK293 cells transfected with rat P2X2 receptors displayed a similar EC50 for ATP that was in the micromolar range (0.75 µM) and therefore too high for reliably detecting low nanomolar concentrations of ATP (see Results section and Fig. 2a). However, we have previously shown that homomeric P2X2 receptors are strongly and selectively potentiated by the steroid/neurosteroid PROG {De Roo, 2010 #102}.

In line with this finding, preincubation of the HEK293-P2X2 cells with PROG increased by sevenfold the sensitivity of the cells to ATP, shifting the EC50 for ATP to 0.1 µM. Moreover, the high oil-water partition coefficient of steroids/neurosteroids such as PROG and PROG derivatives which is >50.000 {Akk, 2005 #65} provides an ideal situation in which these substances can rapidly gain access to the lipophilic compartment of the plasma membrane where they remain trapped for long periods of time. The residence/dwell time of these lipophilic substances within the membrane will be mainly determined by a combination of slow wash out (diffusion into the extracellular medium) and metabolism of the substances within the cell {Akk, 2007 #64}. Once in the hydrophobic part of the membrane bilayer, these steroids can diffuse laterally and interact with integral membrane proteins to modulate their activity, as it has been shown for the positive allosteric modulation and direct activation of GABAA receptors {Akk, 2005 #65;Hosie, 2006 #80;Inquimbert, 2008 #82;Shu, 2004 #79}. A similar effect is probably involved in the potentiation of P2X2 receptors by PROG {De Roo, 2010 #102}.

Whatever the mechanism involved, we noticed that when the HEK293-P2X2 receptors were in contact with PROG, i.e. 5 µM for 100 s or 200 nM for 1 h, we observed a robust potentiation of P2X2 receptor activity that lasted for at least 3 hours after removing PROG from the extracellular solution. We took advantage of this situation and incubated the HEK293-P2X2 cells alone with 200 nM PROG before plating them on top of the cultured DH glial cells, thus avoiding to expose the glial cells directly to PROG. This system allowed us to reliably detect ATP release from the DH glial cells (see below) but also from neurons such as primary sensory neurons (data not shown) (XX add suppl Fig showing ATP release from DRG after High K and absence of High K on HEK cells alone). More generally, we think that this detection system could be useful for the study of ATP release from virtually every cell type.

Vesicular ATP release from cultured DH glial cells and its modulation by NA

The improved sniffer cells system described above allowed us to clearly detect NA-evoked ATP release from DH glial cells. Application of NA or the specific α1 adrenoceptor agonist PHE induced peak-like [Ca 2+ ]i transients in the detector HEK293-P2X2-PROG cells. These transients were absent when the DH glial cells were loaded with the calcium chelator BAPTA-AM or when the extracellular medium contained PPADS, an antagonist of P2X2 receptors.

These results indicated that activation of α1 adrenoceptors in DH glial cells induced calciumdependent release of ATP that was detected by the HEK293-P2X2-PROG cells. This calciumdependent release apparently resembled that described for vesicular release of ATP, glutamate, aspartate or D-serine from astrocytes in the CNS {Gundersen, 2015 #83;Harada, 2015 #84}. In line with these studies, ATP release induced by PHE from our cultured DH glial cells was completely blocked when the DH glial cells were pretreated with bafilomycin A1, an inhibitor of the proton pump present on the membrane of acidic secretory vesicles, preventing the loading of the vesicles by the transmitter {Bowman, 1988 #66;Yoshimori, 1991 #67}. Most interestingly, calcium-dependent ATP release induced by α1 adrenoceptor stimulation was also blocked when DH glial cells were treated with GPN, a small peptide-like molecule that is selectively taken up by secretory lysosomes and leads to their selective functional perturbation {Haller, 1996 #100;Jadot, 1984 #70;Zhang, 2007 #68}. The complete suppression of PHEinduced ATP release by GPN suggested that α1 adrenoceptor stimulation selectively recruited a pool of secretory lysosomes and induced their exocytosis. This is consistent with previous studies in which it was shown that ATP release from hippocampal and cortical astrocytes seems to involve mainly secretory lysosomes {Jaiswal, 2007 #87;Li, 2008 #69;Verderio, 2012 #85;Zhang, 2007 #68}. However, we observed that a release of ATP independent of α1 adrenoceptor stimulation was still possible after selectively affecting secretory lysosomes with GPN. Indeed, it has been shown that in astrocytes, application of a low K + containing solution induced an increase in [Ca 2+ ]i by activating inwardly rectifying Kir4.1 K + channels {Dallwig, 2000 #98;Hartel, 2007 #63}. In our DH glia cultures, application of a low K + solution increased [Ca 2+ ]i in the vast majority (87%) of cells and this led to the release of ATP that could be detected by the HEK293-P2X2-PROG sniffer cells (see Fig. 5). Like for the PHE effect, low K + -induced ATP release was blocked by BAPTA loading of the glial cells or by pretreating the cells with bafilomycin A1. Yet, pretreatment of the DH glia with GPN did not block the effect of low K + on ATP release, whereas ATP release induced by PHE was completely suppressed under these conditions. This clearly pointed to the existence of a second pool of vesicles mediating calcium-dependent ATP release that was distinct from secretory lysosomes and that could be recruited by application of a low K + solution but neither by NA nor PHE. This pool could correspond to the small clear vesicle pool or the large dense-core vesicle pool that are present in astrocytes {Verderio, 2012 #85}.

Spontaneous release of ATP from DH glial cells

Transiently/temporarily interrupting the continuous superfusion of the cells with extracellular solution allowed us to reveal an increase in [Ca 2+ ]i in approximately half of the sniffer HEK293-P2X2-PROG cells seeded on top of the DH glial cells. This increase in [Ca 2+ ]i was inhibited by PPADS, preloading the glial cells with BAPTA or pretreating them with bafilomycin A1, indicating that this increase in [Ca 2+ ]i reflected the release of ATP from at least a subset of glial cells. The fact that only half of the detector cells displayed such an increase in [Ca 2+ ]i might also reflect that only a fraction of the sniffer cells was appropriately positioned (i.e. close to the release sites of ATP) to detect ATP release. The fraction of responsive cells might therefore be a lower estimate of the fraction of glial cells that are able to release ATP spontaneously. Most importantly, spontaneous ATP release was still present after blocking vesicular ATP release from acidic vesicles by bafilomycin A1 pretreatment but was suppressed when glial cells were preloaded with BAPTA. The study of the exact cellular mechanisms underlying spontaneous ATP release were beyond the main scope of our study and were not further investigated. the concentration of K + was lowered to 0.2 mM (low K + solution) triggered an increase in [Ca 2+ ]i that lasted as long as the solution was applied and then rapidly returned to preapplication baseline levels. This effect of the low K + solution on [Ca 2+ ]i could be repeated at least two times in the same cell. b The proportion of DH glial cells displaying an increase in [Ca 2+ ]i following the application of the low K + solution was not affected in the presence of PPADS (50 µM) in the extracellular medium (88.6%, 39/44, Fisher's exact test, P > 0.05 n.s.) but strongly reduced when the glial cells were preloaded with the calcium chelator BAPTA

Legends to figures

  #3}. Noradrenergic projections from the brainstem have been shown to modulate nociceptive information at the spinal level {Budai, 1998 #6;Pertovaara, 2006 #3}. These descending noradrenergic projections to the DH originate mainly from the Locus Coeruleus (LC) {Lyons, 1989 #5} and are recruited during sustained activity of Aδ primary nociceptors {Hitoto, 1998 #7} or during peripheral inflammation {Tsuruoka, 1999 #35;Tsuruoka, 2012 #36}.

  #12}, decrease the excitability of DH neurons/interneurons {North, 1984 #19;Sonohata, 2004 #12} and facilitate inhibitory (GABAergic and glycinergic) transmission in the DH {Baba, 2000 #9;Baba, 2000 #10;Seibt, 2015 #41}. From a behavioral point of view, NA and α adrenoceptor agonists induce antinociceptive effects when administered intrathecally {Pertovaara, 2006 #3;Yaksh, 1985 #13}.

  since it was blocked by a P2X receptor antagonist (see Results section and Fig 3) and was never observed in HEK293-P2X2-PROG cells cultured alone (i.e. in the absence of glia, data not shown). (XX add suppl fig effect of Phe on HEK cells alone + positive control with ATP)

  K + solution never increased [Ca 2+ ]i in HEK293-P2X2-PROG cells cultured alone (in the absence of glia, data not shown). (XXadd suppl fig absence of effect of low K on Hek cells + positive control ATP)Incubation of cells with substancesIn some of the experiments DH glial cells were pre-incubated with pharmacological substances in order to block increases in[Ca 2+ ]i (BAPTA-AM), release of secretory lysosomes (GPN) or release of acidic vesicles (bafilomycin A1). All incubations were performed at room temperature and the substances were added at their final concentration to the extracellular HEPES-based medium used for calcium imaging experiments. The concentrations and times of incubation were as follows: BAPTA-AM (10 µM, 1h); GPN (100 µM; 20 min) and bafilomycin A1 (0.2 µM; 90 min). Concentration and incubation times for the different substances were chosen according to those used in similar studies and published in the scientific literature (XX give refs for GPN, Baf A1 and BAPTA: BAPTA-AM Innocenti JNeurosci 2000,GPN: Li JNeurosci 2008 et Zhang Nat Cell Biol 2007, BafiloYoshimori JBC 1991)

  including for DH neurons in culture {Dallwig, 2000 #98;Petitjean, 2014 #18}. . (XX include supppl Fig showing absence of effect of High K on glila and effect of ATP) The absence of neurons was most probably due to the purely mechanical dissociation protocol that we used.

  Fig. 1c summarizes the proportions of type 1, 2 and 3 responses observed following the application of NA, PHE, ISO and CLO. Interestingly, in the majority of cases (90.2%, 101/112), the responses triggered by PHE could be reproduced at least 2 times in the same cells when applications were separated by a delay of 1000 s (data not shown). (XX add suppl fig showing reproducibility and histo of quantification of PHE response in glia)

  Fig illustration non fading response for 3h). The effect of PROG was due the positive modulation of P2X2 receptors because ATP receptors mediating the increase in [Ca 2+ ]i were

  vesicles and secretory lysosomes {Bowman, 1988 #66;Yoshimori, 1991 #67}. GPN is a substrate of cathepsin C, a lysosomal peptidase present in lysosomes {Jadot, 1984 #70}. GPN is taken up by secretory lysosomes and following hydrolysis by cathepsin C it generates fragments that accumulate inside the secretory lysosomes and rapidly induces an intralysosomal hypertonic condition that leads to the permeabilization of these secretory lysosomes to low molecular weight substances {Li, 2008 #69;Zhang, 2007 #68}. Incubation with GPN therefore induces a selective functional perturbation of secretory lysosomes {Haller, 1996 #100;Li, 2008 #69;Zhang, 2007 #68}. We first verified that pretreatment with GPN and/or bafilomycin A1 did not directly affect the change in [Ca 2+ ]i triggered by PHE or low K + solution in DH glial cells cultured alone.

  However, in line with literature concerning ATP release from glial cells, the non-vesicular spontaneous release of ATP observed in our model might possibly involve release of ATP through ATP-permeable membrane channels, or membrane transporters {Blum, 2008 #103;Dubyak, 2012 #96;Verkhratsky, 2018 #73}. Physiological considerations ATP release from DH glia. Our results confirm that secretory lysosomes are important for ATP release from DH glial cells. Moreover, this lysosomal pool of ATP is apparently the selective target of NA-mediated regulation of ATP release from these cells. The stimulation of ATP release by NA involved α1 adrenoceptors that are coupled to phospholipase C activation {Verkhratsky, 2018 #73}. Lysosome-mediated ATP release has been shown to underlie propagation of calcium waves in glial networks and glia-to-neuron communication {Verderio, 2012 #85}. Interestingly, another vesicular but non-lysosomal pool of ATP could be released when [Ca 2+ ]i was increased by application of a low K + solution {Dallwig, 2000 #98;Hartel, 2007 #63}. In heterologous expression systems transfected with Kir4.1 channels, a decrease in extracellular K + concentration ([K + ]o) to a final concentration of 2 mM was sufficient to trigger important increases in [Ca 2+ ]i in about 40% of the recorded cells {Hartel, 2007 #63}. Decreases in [K + ]o following neuronal activity (K + undershoot) have been observed in the cortex {Heinemann, 1975 #88}, the hippocampus {D'Ambrosio, 2002 #89} and the spinal cord {Jendelova, 1991 #90}. It remains to be established if the observed decreases in [K + ]o observed are sufficient to trigger rises in [Ca 2+ ]i under physiological conditions. Important reductions in [K + ]o might be achieved in very localized regions where the volume of extracellular space is particularly small. Finally, the spontaneous calcium-dependent and non-vesicular release of ATP is of particular interest. This release might serve homeostatic purposes by locally allowing the production of adenosine {Pascual, 2005 #91} but might also represent an important element for basal and activity-dependent signaling within the glial network and between the glial and neuronal networks. ATP and NA systems in the DH. Stimulation of glial ATP release by NA contained in the descending axons of Locus Coeruleus neurons might participate in the control of nociception {Millan, 2002 #2;Pertovaara, 2006 #3;Seibt, 2015 #41}.ATP is an important extracellular signaling molecule (REFXXBurnstock Physiol Rev 2007) that plays a role in sensory transmission (REFXX Nakatsuka PflugersArch 2006), in particular in nociceptive transmission (REFXXTsuda Brain Res Rev 2010). In the spinal cord, ATP can be released from neurons and glial cells (REFXX Jo NatNeurosci 1999, Scemes ) and receptors for ATP are expressed by both cell types (REFXX REF pour neurons + Fields Burnstock NatRevNeurosci). Different P2X receptor subunits are expressed in the DH (REFXX Collo J Neurosci) and their expression appears to increase during the post-natal period, in particular in the deep DH of the spinal cord (REFXX XX Shiokawa J Physiol 2006). Glial cells play a role in setting basal threshold for mechanical nociception (REFXX Foley MolPain 2011) and ATP release participates in the development of neuropathic pain (REFXX Ji & Nedergaard Pain 2013, Masuda NatComm 2016 release neuronal ATP). Moreover, P2X receptors have been shown to participate in the processing of nociceptive signals in the DH and glial cells seem to be involved in this phenomenon (REFXX Aoyama Pain 2011). The noradrenergic innervation of the DH in due to axons of neurons having their cell bodies in the Locus Coeruleus (REFXX Fritschy Brain Res 1987, Lyons J Neurosci 1989). In the rat, noradrenergic axons are observed in the DH at birth and display a high affinity uptake mechanism for NA that reaches its adult-like characteristics during the third postnatal week (REFXX Singer Brain Res 1980). The development of functional adrenoceptors follows a similar time course although the presence of noradrenergic fibres is apparently not necessary for the pattern of expression of the receptors (REFXX Singer Brain Res 1980). In conclusion, our results show for the first time the coexistence of three distinguishable pools of ATP and mechanisms of ATP release from postnatal DH glial cells. In the DH of the spinal cord, this ATP release and its modulation by NA or other neurotransmitters, might play a pivotal role in the modulation of nociceptive signaling.

Fig. 1

 1 Fig. 1 Effect of noradrenaline (NA) and different adrenoceptor agonists on [Ca 2+ ]i in cultured fura-2-loaded DH glial cells. a Application of NA (20 µM, 50 s) elicited three types of [Ca 2+ ]i responses that consisted of a peak and a plateau (type 1), oscillations in [Ca 2+ ]i (type 2) or a single initial peak (type 3). b Effect of different classical adrenoceptor agonists on [Ca 2+ ]i in

Fig. 2

 2 Fig. 2 Characterization of the ATP biosensor detector system. a Proportion of HEK293 cells

Fig. 4

 4 Fig. 4 Application of a low K + solution induced calcium-dependent ATP release from DH glial

(

  Fisher's exact test, ****P < 0.0001). c When fura-2 loaded HEK293-P2X2-PROG cells were plated on top of DH glial cells, application of a low K + solution triggered an increase in [Ca 2+ ]i indicative of ATP release from DH glial cells. d The mean AUC of the calcium signals measured in detector HEK293-P2X2-PROG cells following the application of the low K + solution was also significantly reduced when P2X2 receptors were blocked in the steady state presence of PPADS (50 µM) (unpaired Student's t-test, ***P < 0.001) or when DH glial cells were preloaded with BAPTA-AM (unpaired Student's t-test, ***P < 0.001). Number of cells was between 37 and 131.

Fig. 5

 5 Fig. 5 Characterization of vesicular ATP release from DH glial cells and its modulation by PHE and low K + . a Comparison of calcium responses in detector HEK293-P2X2-PROG cells plated

Fig. 6

 6 Fig. 6 Identification of a spontaneous calcium-dependent but non-vesicular release of ATP from

We would like to thank Mrs Catherine Moreau and Mrs Chantal Fitterer for excellent technical assistance. This work was supported by the Centre National de la Recherche Scientifique, Université de Strasbourg and the French National Research Agency (ANR) contract ANR-17-EURE-0022. V.E. was the recipient of an Initiative d'Excellence (IdEx) fellowship in the framework of the Investissement d'Avenir program of the Université de Strasbourg.

Compliance with ethical standards

Conflicts of interest The authors declare no conflict of interest Ethical approval All applicable international, national and institutional guidelines for care and use of animals were followed.

P2X2 cells (open circles) indicated an apparent EC50 of 0.75 ± 0.02 µM. When cells were preincubated for 1 h with 200 nM PROG (HEK293-P2X2-PROG, filled circles), the doseresponse curve was shifted to the left (EC50 of 0.10 ± 0.01 µM) reflecting an increased detection capacity at low ATP concentrations. Each point represents mean ± s.e.m.


Julien Balestra
email: julien.balestra@univ-cotedazur.fr

Jean-Luc Berenguer

Florence Bigot-Cormier

Françoise Courboulex

Lucie Rolland

David Ambrois

Martin Van Driel

Philippe Lognonné

The InSight Blind Test: an Opportunity to Bring a Research Dataset into Teaching Programs

. This project was originally designed to prepare scientists for the arrival of real data from the upcoming InSight mission. This paper presents the work carried out by middle and high school students during this challenge. This project offered schools the opportunity to participate in and strengthen the link between secondary schools and universities.

. The schools achieved a total of 15 correct detections over a short period. While this number may seem modest compared with the 239 synthetic marsquakes included in the IBT waveform data, these correct detections were entirely made during class time. All in all, the students seemed to be fully engaged, and this exercise seemed to increase their scientific inquiry skills in order to fulfill their task as a team.

Introduction

The InSight mission includes an education and outreach program (E&O) in each partner country. In France, the E&O is carried out by the Géoazur education team. Many pedagogical resources were proposed for this mission, including aspects from launch to landing. The IBT was an excellent opportunity to prepare students to work on future Martian seismograms. The organizers prepared a synthetic dataset of continuous waveforms and invited participants to detect both tectonic and impact seismicity, along with different sources of noise (Murdoch et al., 2017a, 2017b, Kenda et al., 2017). Note that:

-the IBT stopped on February 2018, -the continuous synthetic signal was considered for a fictional year 2019 (from January 1 to December 31).

All mentions of data from 2019 concern this fictional year.

The objective was to prepare research teams interested in developing detection procedures and assess the quality of their work by comparing the seismicity catalog they produced with the IBT synthetic seismicity catalog.

Given that using data in schools from scientific research has shown a positive impact on students (Zollo et al., 2014, Bigot-Cormier and[START_REF] Florence | How students Can Experience Science and Become Researchers: Tracking MERMAID Floats in the Oceans[END_REF], we asked the IBT organizers if the participation of schools was possible. We proposed having the students determine key event parameters such as date, arrival time of seismic waves, epicentral distance and backazimuth direction, terms used in the French educational curriculum. The organizers accepted this format. The School Team was composed of 14 schools either in France or abroad (Fig. 1), all of which are part of the French educational seismic network [START_REF] Courboulex | SISMOS à l'Ecole : A Worldwide Network of Realtime Seismometers in Schools[END_REF]. Catts Pressoir Middle School is a Haitian school, but their teaching program is similar to that of the French curriculum followed by the other schools. The IBT was considered to be an excellent scientific dataset for teaching lessons and was aligned with a series of expected skills, which will be highlighted throughout this study.

One of the expected skills is to identify, extract and organize information from scientific data.

In middle school lessons, earthquakes are taught as being the result of the Earth's internal activity. Students learn that ground motions during an earthquake are due to different waves shaking the surface. A typical class exercise is to identify different seismic phases in a seismogram. During subsequent high school lessons, a classic exercise is to determine the arrival times of seismic P and S waves in order to locate the epicenter. We were strongly convinced that the data from the IBT could be used instead of these classic exercises as a previously unseen dataset. Furthermore, it was felt that student motivation would be increased by knowing that the InSight science team would analyze their results.

At the start of this project, a training process was required to efficiently prepare students for the upcoming synthetic data analysis.

Student Training Process

Real Earth and Synthetic Mars Data

In order to train students and increase their skills in seismic signal analysis, we provided students with two newsletters in October 2017. Each document was in PDF format, with numerical data attached. In this study, all numerical datasets were analyzed with SeisGram2K80_ECOLE.jar (SG2K80, Lomax A., 2000) software. A specific velocity model was implemented by A. Lomax (from [START_REF] Sohn | The interior structure of Mars: Implications from SNC meteorites[END_REF].

The task approach was to study examples of signal processing. Students first worked on decimation and its effect on seismic data. We provided real seismograms recorded at the BLOR education station ("Lycée de la Montagne" high school in Valdeblore, France). The earthquakes considered were:

the April 7, 2014, Barcelonnette earthquake (Mw 4.9, France, 0.6° epicentral distance); -the April 16, 2016, earthquake (Mw 7.8, Ecuador region, 87.6° epicentral distance).

Students then worked on bandpass filtering and frequency content. We provided synthetic signals from the IBT. The proposed fictional time periods were:

-January 10 to 15, 2019; -July 15, 2019; -September 22, 2019.

We rotated these signals into the north, east, and Z (vertical) directions from metadata provided by the IBT organizers using the ObsPy packages [START_REF] Krischer | ObsPy: a bridge for seismology into the scientific Python ecosystem[END_REF]. No instrumental correction for response was applied.

Decimation of Seismic Signals: an Exercise to Manipulate, Experiment, and Understand the

Nature of the Data Used

The first exercise was designed to help students become familiar with the nature of the IBT dataset, especially with the difficulty of working with decimated seismic data. The SEIS seismometer stores data at 100 Hz. At the time of the IBT, the initial sampling rate retained for future real data transmission was 2 Hz. This corresponds to a volume of data transfer guaranteed by the NASA. The educational seismic stations used by students typically store ground motion at 50 Hz. Therefore, the first exercise was to introduce the synthetic data corresponding to decimated data, i.e. with lower resolution than the raw data. Students worked on the Barcelonnette earthquake decimated to 2 Hz. Decimation was carried out using the Python ObsPy software package (with an anti-aliasing low-pass filter). Figure 2a shows raw and decimated seismograms (vertical component). Change in amplitude was the first effect observed by students. They understood that samples were missing, which implies that information about ground motion was missing. Zooming in with SG2K80 also allowed them to observe changes in shape (Fig. 2b). After these first manipulations, students worked on the teleseismic event. At first they observed no significant changes in amplitude between the raw and the decimated signal (Fig. 2c). They observed that the decimation had very little effect on the signal. Zooming in enabled them to observe small differences due to the anti-aliasing prefiltering (Fig. 2d).

This first analysis of changes of seismogram shapes enabled students to understand the effect of this kind of processing on raw data. Understanding the link between the content of a dataset, and the information that can be extracted from it, is an important skill required in French educational programs. Moreover, students were also able to imagine how difficult it would be to analyze future real Martian seismograms decimated to 2 Hz. This first step concluded with the introduction of the relationship between the shape and period of the signal. Signals were described as being composed of tighter or looser arcs, with tight arcs corresponding to short periods and broad arcs corresponding to long periods, following the approach of [START_REF] Florence | How students Can Experience Science and Become Researchers: Tracking MERMAID Floats in the Oceans[END_REF]. This allowed us to introduce a second step based on frequency analysis.

Bandpass Filter and Spectrogram: Use of Digital Tools to Identify Seismic Waves

The frequency content of seismograms was introduced in response to the question raised by students: "How will scientists be able to detect seismic activity?". For both middle and high school students, the use of a bandpass filter is unusual. Understanding the calculation of spectrograms is too difficult, and it is not a subject in the French curriculum. However, students are expected to use digital processing software. SG2K80 proposes tools to filter seismograms and to compute a corresponding spectrogram, which was introduced as a data processing method to highlight the frequency content of the continuous signals. To become familiar with these different aspects, students were invited to work with the synthetic seismogram on January 12, 2019 (IBT fictional day, vertical component, Fig. 3). Note that any mentions below of a synthetic seismogram refer to the IBT, except seismograms computed in the study of [START_REF] Bozdağ | Simulations of Seismic Wave Propagation on Mars[END_REF].

Students started this new activity by applying bandpass filters to understand their effects on the seismograms analyzed. Two frequency intervals were provided:

from 0.001 Hz to 0.01 Hz (lf-bp for low frequencies bandpass), -from 0.01 Hz to 1.0 Hz (hf-bp for high frequencies bandpass).

The frequency of 1 Hz corresponds to the Nyquist frequency, i.e. the maximum frequency that can be analyzed for a decimation to 2 Hz. The comparison of these three signals (raw and filtered) allowed students to highlight different points of the filtering process. By applying lfbp filtering, they observed that the very long period arc observed in Figure 3a was filtered out.

They also observed a long duration event (Fig. 3b). With hf-bp filtering this long duration event was always observed along with a later and shorter duration event (Fig. 3c).

This kind of processing is consistent with the skills required by the educational curriculum.

Students showed they understood that:

events (seismic, atmospheric, etc.) observed in a seismogram have their own frequency characteristics;

the filtering process is used to try to highlight expected or searched events in seismograms;

the shape of seismograms varies according to removed frequencies.

Although, in this case, a filtering process allows the detection of significant events, we invited students to analyze the computed spectrogram from the raw seismogram (SG2K80 spectrogram tool, Fig. 3d and3e). Students observed that the long and later short events are easier to detect by displaying the frequency content of the seismogram reading. Figure 3d shows that the two events are highlighted by an increased amplitude for specific frequencies (about 0.1 Hz for the long event, up to 0.8 Hz for the shorter event), which indicates a significant change in ambient ground motion.

Students understood that hidden synthetic marsquakes could be easier to detect by processing data. To go even deeper into the analysis, zooming in on the late and short event was proposed (Fig. 3e). Students observed that the long duration signal is composed of a frequency content different from the shorter duration event. They also observed an extended coma-like shape (Fig. 3e, black dashed ellipse). From discussions with seismologists at Géoazur (who also participated in the IBT as one of the challengers, [START_REF] Van Driel | Preparing for InSight: Evaluation of the Blind Test for Martian Seismicity[END_REF], this specific shape was designated as the signature of Rayleigh waves. These surface waves have a velocity that is dependent on their frequencies. Thus, some Rayleigh waves arrive earlier and some arrive later, which reflects their dispersion. This specific shape was used to determine and identify them.

The frequency content of body waves was also approached. From the July 15 (M4.3) and the September 22 (M5.0) synthetic events, the frequency content of body waves was considered as ranging from 0.3 Hz to 0.9 Hz (higher than the frequency content of Rayleigh waves). The work on seismic phases with these large synthetic events was considered as sufficient to help students to detect smaller nearby quakes with higher frequency content.

The training process stopped here for middle school students. According to their educational curriculum, they were ready to analyze synthetic data. Their goal was to identify arrival times of seismic waves and to propose their part of the School Team catalog of seismicity. It was possible to extend this analysis for high school students, thus the second newsletter was given to them.

Guided Analysis and Interpretation of Synthetic Signals from the InSight Blind Test: Skills Expected for High School Students

A. Estimation of the epicentral distance based on Rayleigh wave arrival times

Extracting information from scientific datasets is an expected skill for high school students.

Locating the epicenter is a good exercise for this, and even more so if the dataset comes from a current scientific project. Typically, seismic events are located by analyzing P and S wave arrival times from three or more seismic stations. One aim of the second newsletter given to students was to introduce a location technique using only one three-component station, without any knowledge of an accurate deep structure of the planet, and without the origin time. We proposed using successive Rayleigh wave arrival times [START_REF] Panning | Verifying single-station seismic approaches using Earth-based data: Preparation for data return from the InSight mission to Mars[END_REF]. Hereafter this paper will use the following notations (Fig. 4a and4b):

-t1: the first arrival time of Rayleigh waves at the virtual station, i.e. the shortest surface travel time between the epicenter and the station (LR1, for Long-period Rayleigh 1); -t2: the second arrival time of Rayleigh waves at the station, i.e. the longest surface travel time between the epicenter and the station (LR2); -t3: the third arrival time of Rayleigh waves at the station, i.e. the shortest surface travel time between the epicenter and the station, plus a complete surface trip around the planet (LR3).

The method from [START_REF] Roques | A single geophone to locate seismic events on Mars[END_REF] was used to illustrate this approach (Fig. S1). The synthetic seismograms used in this method came from the study by [START_REF] Bozdağ | Simulations of Seismic Wave Propagation on Mars[END_REF]. They were computed at virtual stations along the Mars equator, spaced by 20°. The mathematical formula to compute epicentral distance from t1, t2, and t3 arrival times is:

𝐸𝑝𝑖𝑐𝑒𝑛𝑡𝑟𝑎𝑙 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 = 𝑡3-𝑡2 𝑡3-𝑡1 * 𝜋 * 𝑅 𝑝𝑙𝑎𝑛𝑒𝑡 (1)
where 𝑅 𝑝𝑙𝑎𝑛𝑒𝑡 is the radius of the considered planet. Distances are in kilometers and arrival times are in seconds. This work was interesting because distances and origin times were known.

Students were easily able to confirm their results.

We then invited students to work with an unknown event from the IBT using the synthetic seismogram from September 22, 2019 (Fig. 4b). We chose this event because the analysis of its frequency content showed specific signatures considered as a marker of Rayleigh waves.

Students were able to observe the different wave trains in the time domain, which presented specific signatures in the frequency domain. From this analysis and by picking the three passages of Rayleigh waves, students estimated an epicenter located at 35.5° from the station.

Thus they understood that this approach is not enough to locate the event. They estimated a distance, but the direction was missing. This aspect was the subject of the last training exercise.

B. Azimuth and back-azimuth estimation from the rotation of horizontal components

This last exercise was introduced using a simple hands-on activity. We provided electronic accelerometers and the RISSC© (Record Interface Sensors at School, see Data and Resources)

interface. This educational interface displays records from each component in real time, which are identified with a sticky label (Fig. S2). The accelerometers were fixed on a table, and students followed two procedures: first, to apply an impact parallel to the table plane in the X direction of the device, and second, to apply an impact in the Y direction of the device. Using records displayed with RISSC, students observed the difference in amplitude of each component and concluded that the maximum amplitude is observed in the main direction of wave propagation. In class, students then reviewed the relationship between azimuth and backazimuth. SG2K80 software includes a tool to compute the angle value of the azimuth from the first P wave amplitude on horizontal components (Fig. 5). This function allows recomputed signals to be displayed after a chosen rotation value (as a virtual rotation of the sensor in the geographical coordinate system). Figure 5a shows the first P wave on each component, without rotation. Figure 5b shows a flat P wave on the East component for a 65° (clockwise) rotation. This is also the value for which the P wave amplitude is maximal on the North component.

Students understood that this virtual rotation of the sensor allows them to determine the direction for which the first ground motion is maximal on one of the horizontal components and zero on the other.

The last training exercise involved determining the back-azimuth. From the P wave polarity Blind Test Independent Data Analysis and the Creation of the School Team Catalog

The analysis phase started at the beginning of November, 2017, and ended in January, 2018.

This period was long enough for teachers to integrate this challenge (training and analysis phases) into their official teaching hours. Students worked for six to ten hours on this project.

We now describe two activities performed by students at each school.

A Daily Atmospheric Signature

During the one-year-long synthetic seismogram, disturbances in the continuous signal were not exclusively due to traveling seismic waves. For example, environmental noise [START_REF] Spiga | Structure and dynamics of the convective boundary layer on mars as inferred from large-eddy simulations and remotesensing measurements[END_REF]) was added in order to create a signal that was as realistic as possible [START_REF] Clinton | Preparing for InSight: An Invitation to Participate in a Blind Test for Martian Seismicity[END_REF], with the associated modeled seismic noise originating from the interaction of the environment with the lander or the ground (Murdoch et al, 2017a, 2017b, Kenda et al., 2017). See [START_REF] Spiga | Atmospheric Science with InSight[END_REF] for a general review of atmospheric seismic noise and [START_REF] Lognonné | SEIS: Insight's Seismic Experiment for Internal Structure of Mars[END_REF] for noise shielding on the SEIS instrument.

During the training steps, students identified an unknown long event in the synthetic seismogram that occurred on January 12, 2019 (fictional day from the IBT). During the analysis phase, students observed that in fact this long event occurred each day, but not at the same hour.

Students asked us for an explanation of this phenomenon, and we submitted the question to a researcher, who gave them the following explanation. The sun warms the surface of Mars, which causes thermal agitation (convection) near the surface. At nightfall, the surface cools because the sun no longer warms it and convection stops. The wind blows, but there are no rapid turbulent fluctuations that produce this atmospheric noise. The observed time lag can be explained by the length of Martian days, which last approximately 24 hours and 39 minutes.

One of our aims was reached through this interaction, i.e. to create a link between students and researchers, and to improve the students' scientific inquiry skills.

Estimation of the Epicentral Distance of an Unknown Event That Occurred on July 15, 2019

This section presents the analysis provided by high school students of the event of July 15, 2019 (fictional day from the IBT). They were able to observe different wave trains on the vertical component and they clearly identified two Rayleigh wave signatures in the frequency domain (Fig. 7). However, without a clear third signature from the whole day's seismogram, they decided to pick a third Rayleigh wave at the start of an increase of the scale amplitude around 7:00 a.m. By picking these three Rayleigh waves, they obtained an epicentral distance equal to 90.5° (from the corresponding SG2K80 tool). The correct epicentral distance was 90.94°. The slight change in amplitude in the frequency domain is probably due to the start of daily atmospheric disturbances, but the lower value of the bandpass filter value applied by students (0.1 Hz to 1 Hz, Fig. 7) was too high to highlight this daily event. However, we appreciated their approach and their thinking through the use of the analysis processes they learned during the training phase.

These many false detections could be the starting point for a new educational project with a dataset from the IBT. Future student groups could start analyzing why these false detections were made. The aim would be to improve the training phase documentation in order to facilitate the dismissal of some events in the continuous signal.

This study could be conducted once again at middle schools and high schools, which could be paired. Middle school students would work on identifying seismic waves, and the high school students would have two objectives: i) validate or invalidate detections, ii) try to estimate a location for detections considered as true. This networking would allow for an increase of educational skills. Furthermore, annual educational projects are planned in new educational programs for high schools. A longer period to work on this challenge would be useful to enable us to compare the quality of the catalog provided.

Conclusion

Of course, our ambition was not to compete with other science teams. Our main goal was to highlight the IBT dataset to school students. This objective was achieved because of motivated teachers who decided to engage their students in this challenge. No written evaluation was specifically carried out, but all teachers reported their satisfaction with the work provided by students. They were also satisfied that they had been able to include this dataset in their own activities. Figures S5a andS5b show pictures of students working on this challenge. Even the few students with learning difficulties responded well to the project, showing a good level of involvement and were engaged in class discussions. We think that the reason was that the framework was out of the ordinary. The aim was not to work on a typical exercise, but to suggest a catalog to the InSight Science Team.

Teacher feedback focused on the great impact of the IBT on classroom dynamics. It allowed students to develop the following main skills:

practice a scientific approach;

demonstrate observation skills, curiosity, critical thinking;

experience autonomy;

communicate in scientifically appropriate language: oral, written, graphic, numerical.

Middle school students showed that they were able to detect synthetic events, even though their scientific background was less developed than that of high school students. This point highlights that involvement and seriousness, and not the age of students, were the main determining aspects to properly carry out this challenge.

The French EduMed Observatory educational project organized a seminar in the French Géoazur laboratory. Students from high schools came to present the classwork they had carried out during their school year. One group presented their work from the September 22 synthetic quake (Fig. S5c). They presented their picks of Rayleigh waves and their estimate of the epicentral location. They also presented their work on 1D velocity models proposed for the IBT [START_REF] Clinton | Preparing for InSight: An Invitation to Participate in a Blind Test for Martian Seismicity[END_REF]. They estimated the origin time from t1, t2, and t3, and then they calculated the velocity of the first P wave. They also considered the following starting models: i) a homogeneous planet, ii) the source located at the surface, iii) a seismic ray as a straight line between the source and the station. They also computed a theoretical depth reached by this first "straight" P wave. By comparing their results with velocity models published in Clinton et al.

(2017), they understood that they had chosen a wrong starting velocity model, and learned about how seismic datasets are used to understand the deep structure of Mars. Although they did not succeed in determining the model that was used for the IBT, they showed that they developed many expected skills by working on this dataset.

Student teams are now ready and looking forward to analyzing real data from Mars. In addition, educators should keep in mind that even more challenging seismic signals are expected to be recorded on Mars, with a majority of small, nearby marsquakes (higher frequency content, lower signal noise ratio). A couple of teleseismic events will hopefully reveal the deep interior of the planet.

Tables

(

  upwards) on the vertical component, students calculated a back-azimuth equal to 245°. The final catalog of the IBT was not available at the moment of this training process. Students waited for the publication of the solution to evaluate their first detection. FigureS3shows the results of this training exercise validated after the publication of the true catalog. They used the EduCarte-Mars geographical information system (GIS) to display the results of location. The specific Mars digital field model was implemented by A. Lomax. This last activity completed the schools' training for the IBT. The fourteen schools then each received one month of non-overlapping synthetic data and started their analyses during their teaching sessions.

  inverted triangle: seismic station. b) Raw synthetic seismogram and corresponding spectrogram on September 22, 2019 (vertical component). This picture comes from a screenshot with SG2K80. LR1, LR2 and LR3: pick of the Rayleigh wave passage at the station. t1, t2, t3: corresponding arrival times.

Figure 5 .

 5 Figure 5. Back-azimuth estimation for the event on September 22, 2019. (a) An enlargement of

Figure 6 .

 6 Figure 6. Synthetic seismograms from January 10 to January 15, 2019, compiled by a group of

Figure 7 .FiguresFigure 1 .

 71 Figure 7. Study of the unknown event detected by students on July 15, 2019. Raw seismogram

Figure 3 .

 3 Figure 3. Synthetic marsquake on January 12, 2019 (vertical component). (a) Raw seismogram.

Figure 4 .

 4 Figure 4. Elements for the epicenter location from Rayleigh waves. a) Scheme of the three

Figure 6 .

 6 Figure 6. Synthetic seismograms from January 10 to January 15, 2019, compiled by a group of

Figure 7 .

 7 Figure 7. Study of the unknown event detected by students on July 15, 2019. Raw seismogram

Acknowledgments

We are especially grateful to the IBT organizers for accepting a School Team in their challenge and to have provided their dataset. We are also grateful for the help from our scientist partners: Anne Deschamps, Fabrice Peix, Jérôme Chèze (Laboratoire Géoazur), and Aymeric Spiga (Laboratoire de Météorologie Dynamique) for their help and support. InSight Education activities are supported by CNES. This is InSight contribution number: ICN 97. We also are grateful to the reviewers, and especially to Jennifer Hecker for reviewing the English language and grammar used.

Data and Resources

Seismograms from the two earthquakes recorded at the BLOR educational seismic station, SeisGram2K80_ECOLE.jar software, and the RISSC interface are available on the EduMed Observatory website:

the Barcelonnette earthquake: http://edumed.unice.fr/fr/data-center/seismo/donnees-seismo/2014-04-07-5_0_barcelonnette -The Ecuador earthquake: http://edumed.unice.fr/fr/data-center/seismo/donnees-seismo/2016-04-16-7_8_ecuador -SG2K80: http://edumed.unice.fr/fr/contents/news/tools-lab/SeisGram2K -Record Interface Sensors at School (developed by David Ambrois): http://edumed.unice.fr/fr/contents/news/tools-lab/RISSC EduMed Observatory is funded by the University of Côte d'Azur -JEDI Investments in the Future project managed under reference number ANR-15-IDEX-01. Synthetic seismograms for the IBT are available from the specific ETH website (http://blindtest.mars.ethz.ch). The synthetic data presented in this study and EduCarte software (Mars version) are also available on the French InSight educational website supported by the Centre National d'Etudes Spatiales (CNES): -InSight Blind Test dataset: https://insight.oca.eu/images/InSight_Medias/zip/blindtest_daily_synthetic_data.zip -Synthetic data from Bozdağ et al. (2017): https://insight.oca.eu/images/InSight_Medias/zip/data/data-bozdag-2017.zip -EduCarte Mars (© A. Lomax and J.L. Berenguer): https://insight.oca.eu/images/InSight_Medias/zip/software/Educarte-Mars-3.3.0X18.zip

Discussion

The teachers' geophysical skills enabled the project to be properly completed. They have attended various workshops on the subject during their careers and have already worked on educational projects based on seismic data. The teachers included this challenge in their teaching time and at their convenience. As mentioned above, the students worked at most about ten hours. The schools worked independently during the analysis phase. The main reason for this was that teachers chose the allocated period to work on synthetic data. At the end of January 2018, the catalog from the School Team was provided to the IBT organizers. This catalog (Table 1) was then compiled in [START_REF] Van Driel | Preparing for InSight: Evaluation of the Blind Test for Martian Seismicity[END_REF] (Fig. S4). The School Team catalog contained fifteen correct events: thirteen quakes and two impacts. Six high schools and two middle schools found these events. The other schools gave wrong detections. For the students, the main constraints were the available tools and the limited time to work on this challenge.

Detected events were located between 700 km and 8400 km from the chosen seismometer location. The detected synthetic events had magnitudes ranging from 2.5 to 5. The closest event in the true catalog (191 km, M2.5) was not detected. Two events of magnitude 2.5 and 2.6 were detected. Their epicentral locations were 724 km and 713 km respectively. Seven events, ranging from magnitude 3 to magnitude 4, were detected. Their epicentral locations ranged from 1000 km to 6500 km. One event with a magnitude higher than 4 was detected (at 5379 km). The larger event (M5.0, 2000 km) occurred on September 22 (studied during the training process) was also added to the catalog. Two impacts were also detected, the stronger impact on October 24, and a weaker impact on October 25.

In total, 103 events were compiled in the School Team catalog. Changes in the amplitude and frequency of the continuous signal often were considered as seismic waves, because the corresponding computing spectrogram showed changes in amplitudes. These false detections could be a result of the very short teaching time allowed in the training and analysis phases.

List of Figure Captions