Proteoglycans involved in bidirectional communication between mast cells and hippocampal neurons

RESEARCH Open Access Proteoglycans involved in bidirectional communication between mast cells and hippocampal neurons Juan Antonio Flores 1 , María Pilar Ramírez-Ponce 1 , María Ángeles Montes 1 , Santiago Balseiro-Gómez 1,2 , Jorge Acosta 1 , Guillermo Álvarez de Toledo 1 and Eva Alés 1* Abstract Background: Mast cells (MCs) in the brain can respond to environmental cues and relay signals to neurons that may directly influence neuronal electrical activity, calcium signaling, and neurotransmission. MCs also express receptors for neurotransmitters and consequently can be activated by them. Here, we developed a coculture model of peritoneal MCs, incubated together with dissociated hippocampal neurons for the study of cellular mechanisms involved in the mast cell-neuron interactions. Methods: Calcium imaging was used to simultaneously record changes in intracellular calcium [Ca 2+ ] i in neurons and MCs. To provide insight into the contribution of MCs on neurotransmitter release in rat hippocampal neurons, we used analysis of FM dye release, evoked by a cocktail of mediators from MCs stimulated by heat. Results: Bidirectional communication is set up between MCs and hippocampal neurons. Neuronal depolarization caused intracellular calcium [Ca 2+ ] i oscillations in MCs that produced a quick response in neurons. Furthermore, activation of MCs with antigen or the secretagogue compound 48/80 also resulted in a neuronal [Ca 2+ ] i response. Moreover, local application onto neurons of the MC mediator cocktail elicited Ca 2+ transients and a synaptic release associated with FM dye destaining. Neuronal response was partially blocked by D-APV, a N-methyl-D-aspartate receptor (NMDAR) antagonist, and was inhibited when the cocktail was pre-digested with chondroitinase ABC, which induces enzymatic removal of proteoglycans of chondroitin sulfate (CS). Conclusions: MC-hippocampal neuron interaction affects neuronal [Ca 2+ ] i and exocytosis signaling through a NMDAR-dependent mechanism. Keywords: Ca 2+ imaging, neuro-immune, hippocampal neurotransmission, NMDA receptors, proteoglycans, exocytosis Background Communication between the immune and the nervous system is considered an important biological process [1, 2]. While microglia are the resident immune cells in the brain and play a pivotal role in immune surveillance in the central nervous system (CNS) [3,4], the roles of other immune cells remain unexplored. Mast cells (MCs) are also resident in the brain of all mammalian species studied [5] and located in the meninges and perivascular area on the brain side of the blood-brain barrier [6] and the parenchyma of the thalamic and hippocampal regions [7,8]. A key feature of MCs is that they contain in their cytoplasm numerous secretory granules that hold a vast array of inflammatory mediators and biologically active substances such as histamine, serotonin, proteoglycans, and proteases. When appropriately activated, MCs undergo degranulation, a process by which the preformed granule compounds are rapidly released into their surroundings [9]. MC degranulation can occur in response to various external stimuli including IgE receptor crosslinking, complement activation, neuropeptides, and certain toxins [10]. MCs are also © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: [email protected] 1 Dpto. de Fisiología Médica y Biofísica, Facultad de Medicina, Universidad de Sevilla, Av. Sánchez Pizjuán 4, 41009 Sevilla, Spain Full list of author information is available at the end of the article Flores et al. Journal of Neuroinflammation (2019) 16:107 https://doi.org/10.1186/s12974-019-1504-6 temperature and pressure sensitive, degranulating in response to noxious physical stimuli through a mechanism dependent on transient-receptor-potential channel 2 [11]. In addition to inducing the release of preformed granule constituents, MC activation leads to de novo synthesis of many bioactive compounds, including lipid mediators such as leukotrienes or prostaglandins or a variety of cytokines, chemokines, and growth factors [10, 12]. Overall, preformed granular material is released in minutes, while de novo materials are secreted within hours.Notably, MC activation does not necessarily lead to degranulation. For example, exposure of MCs to lipopolysaccharide can cause the release of cytokines without observable degranulation [13]. The mast cell-nerve relationship has been discussed extensively [14–16]. MCs are often found in close proximity to nerves [17]. When stimulated, messenger molecules released from nerves (e.g., neuropeptides) can elicit MC degranulation, but also mast cell-derived mediators (e.g., histamine, serotonin) can modulate neurotransmission [18], creating a bidirectional positive feedback-loop that may result in neurogenic inflammation [19]. MCs and their mediators are important conveyors of the communication between the innate enteric immune system and the enteric nervous system, and there is strong evidence for nerve-MC interaction, particularly in gastrointestinal diseases such as inflammatory bowel syndrome [20,21]. The functions of MCs in the brain are unknown. However, clear evidence indicates brain MCs are important contributors to neuropsychiatric states such as anxiety [22] and are able to modulate neuroinflammation, which may affect diseases such as multiple sclerosis and Alzheimer’s disease [2,23]. The interaction between MCs and CNS neurons has been little studied. We examined the possibility of a bidirectional communication between hippocampal neurons and MCs through the use of a coculture model, where rat peritoneal mast cells, representative of connective tissue mast cells [24], are incubated with dissociated hippocampal neurons. A similar model was developed for the study of the cellular mechanisms involved in the associations between MCs and nerves in vivo [17] and MCs and sympathetic neurons [25,26]. Hippocampal neurons were activated by depolarization and MCs were immunologically activated (by antigen) as well as by the compound 48/80 and LPS. After selective stimulation of MCs and neurons, we used [Ca 2+ ] i imaging to record subsequent cell activation. Besides, a cocktail of MC secretory products was applied directly onto neuronal somata causing [Ca 2+ ] i transients and exocytosis. This response was NMDAR-dependent. We show for the first time bidirectional communication between hippocampal neurons and MCs and a role of MCs in neurotransmission. Methods Preparation of neuronal cultures Hippocampal neurons were cultured from brains of P0– P1 newborn rats isolated by surgical separation of the cranial bones by natural suture. CA-1 and CA-3 hippocampal regions from both sides were isolated under a light surgical microscope and removed from the brain. The two hippocampi were immersed in 1 ml of enzymatic solution and kept under continuous agitation (80 rpm) for 40 min and centrifuged at 200×gfor 5 min. The enzymatic solution contained 1 ml of DMEM (PAA Laboratories), 0.2 mg of cysteine, 1 mM CaCl 2 , 0.5 mM EDTA and 5 units/ml papain (Worthington). The enzymatic solution was previously bubbled with 95% O 2 and 5% CO 2 for 20 min. After centrifugation, the supernatant was removed, and 1 ml of inactivating solution was added to the tissue to prevent further digestion. The inactivating solution contained 800 μl of DMEM (Gibco), 200 μl FBS (BioWest), 200 units of penicillin (Gibco), 200 μg of streptomycin (Gibco), 2.5 mg albumin and 2.5 mg trypsin inhibitor (Sigma). After this process, the tube was centrifuged again at 200×gfor 5 min and mechanically dispersed afterward with a 1-ml pipette tip five times. The supernatant was discarded and replaced with neuronal media, containing: 1 ml Neurobasal (Gibco), 20 μl B27 (Gibco) (50×), 10 μl Glutamax (Gibco) (100×), 20 units penicillin and 20 μg streptomycin. Neurons were plated at a density of 150,000 cells/well (12-well plates) over a single feeding layer of astrocytes grown on 15 mm ϕcoverslips. For the astrocyte feeding layer, the coverslips were previously treated with 1 ml of an acidic solution containing 17 mM acetic acid, 0.8 mg collagen (BD Biosciences), and 1 mg poly-D-lysine (Sigma). For the treatment, the solution was gently spread over the coverslips with a cotton bud and let to dry overnight and washed with water three times. The feeding layer was made of astrocytes from rat and seeded on the cover slips at a density of 40,000 cells/dwell for 5 days. The astrocyte feeding layer was preserved by adding uridine solution that prevents cellular division. The uridine solution contained DMEM, 20.4 mM uridine (Sigma) and 8.1 mM 5-phluoro-2-deoxy-uridine (Sigma). Neurons were cultured between 14–17 DIV in an incubator at 37 ° Cwithanatmosphereof95%O 2 and 5% CO 2 . Mast cell isolation and preparation of cocultures MCs were isolated from the peritoneal cavity of 3- to 5-month-old rats following a procedure described in detail elsewhere [27]. The peritoneal wash fluid contains a large number of cells, a small fraction of which are MCs. Using a Percoll (Sigma-Aldrich) gradient, fully differentiated connective tissue peritoneal MCs were separated from other cells [28]. Freshly purified peritoneal MCs were added to the neuronal cultures at 3 × 10 4 cells per Flores et al. Journal of Neuroinflammation (2019) 16:107 Page 2 of 13 well 18–24 h before the experiment. To induce antigen stimulation, 4 h after seeding, peritoneal MC were sensitized with mouse monoclonal anti-DNP IgE (1 μg/ml). All reactives and drugs were purchased from Sigma, unless otherwise stated. Mediator cocktail released in response to heat 1×10 6 purified MCs obtained after Percoll purification were sedimented by centrifugation (200×g, 5 min) and resuspended in 1 ml of basal Locke solution. The basal Locke solution contained 140 mM NaCl, 10 mM HEPES, 3 mM KOH, 2 mM CaCl 2 , 1 mM MgCl 2 , and 10 mM glucose. The pH of the solution was adjusted to 7.3 with NaOH. Cells were placed into a prewarmed Thermomixer (Eppendorf) for 1 h at 53 °C with gentle agitation. MC degranulation was checked by visual inspection under a microscope. Cells were separated from supernatants by centrifugation at 200×gfor 5 min. Supernatants were aliquoted (50 μl) and stored at −80 °C until use. Recordings of [Ca 2+ ] i signal Changes in [Ca 2+ ] i were monitored by dual excitation microfluorimetry in isolated hippocampal neurons or in cocultured MC-hippocampal neurons incubated in Locke solution containing Fura-2 AM (Molecular Probes) (5 μM for 45 min at 37 °C in the dark) and pluronic F-127 (Sigma) (final concentration 0.004%). Then, cells were washed twice in Locke external solution without the probe and used for imaging. The coverslip with cells loaded with Fura-2 AM was then mounted in a RC-25F perfusion chamber (Warner instruments) and placed on an inverted microscope (AxioVert 200, Zeiss) equipped with a standard filter set (XF04-2; Omega Optical). During recordings, cells were excited by a xenon light source at 360/380-nm wavelengths (exposure time, 0.5 s; data acquisition at 0.33 Hz) by means of two narrow beam band-pass filters selected by a computer-controlled wheel. The emitted fluorescence was filtered through a 520-nm filter, captured with an ORCA-R2 CCD camera (Hamamatsu Photonics). Data were acquired and stored using HCImage software and exported to Igor Pro (WaveMetrics) for analysis. All values were normalized to the basal fluorescence (baseline). The Ca 2+i signal was expressed by the ratio of fluorescence at 360 nm and 380 nm. All experiments were performed at 37 °C. Imaging and analysis of FM dye release Fifteen-millimeter cover glasses with cultured hippocampal neurons (days in vitro (DIV) 12–16) were mounted in the perfusion chamber. Synaptic vesicles were loaded by substitution of basal Locke solution with 70 mM [K + ] Locke solution (generated by equimolar replacement of NaCl by KCl) containing 4 μM FM1-43 (Molecular Probes) for 2 min. After washing in a basal Locke external solution for 10 min, synaptic release from selected neurons was induced by applying 70 mM [K + ] Locke solution or MCM through a pressure pulse (5 s) with a micropipette (1 μmϕ) positioned ~ 100 μm to the neuronal soma. Images were obtained using a 63× Plan- Neofluar (NA 1.3) oil immersion lens (Zeiss) on an Axiovert 200 inverted microscope equipped with a standard filter set for FM1-43 dye (XF115-2; Omega Optical). Images were captured with an ORCA-R2 CCD camera (Hamamatsu Photonics) controlled by HCImage software (Hamamatsu Photonics). Time-lapse images were acquired at a rate of 0.5 Hz, 1344 × 1024 pixels (binned 1 × 1), with an exposure time of 200 ms. Recordings were performed at 37 °C. For quantification of FM dye destaining, circular regions of interest (ROI, 5 × 5 pixels) were located manually over the center of FM dye fluorescence of spatially separated synapses. Raw destaining trace values were extracted and loaded into Igor Pro (Wavemetrics) for further analysis. The destaining traces were graphed as the change in fluorescence (ΔF, absolute values) normalized to the fluorescence at the start of the experiment (F0) using the following equation: ΔF/F0 = (FF −F0)/F0, where FF is the point at which the destaining curve slope becomes zero. The FM dye release curves of single synapses were fitted mono-exponentially. A successful mono-exponential fit was defined by positive values for amplitude (ΔF) and time constant (τ). Statistical analysis Differences between groups were tested for statistical significance using Student’sttest for unpaired data when the data passed a normality test (Lilliefors-corrected Kolmogorov–Smirnov) and a Mann–Whitney Utest when it did not [29]. The data were regarded as statistically different when P< 0.05. All statistical analyses were performed using SPSS Statistics 22 software (IBM). All data were plotted as the mean ± SEM. The presented data represent experiments performed with cells from at least three different cultures. Results Bidirectional communication between hippocampal neurons and MCs in coculture When in a coculture of mixed hippocampal neurons and MCs, loaded with FURA-2 AM, we stimulated a neuron by direct depolarizing stimuli applying a pressure pulse of K + -enriched solution (70 mM KCl with iso-osmotic reduction of NaCl) (Fig. 1a), a high [Ca 2+ ] i transient was evoked upon the onset of the stimulus in neuronal cell body, as it was expected (Fig. 1b). The peak amplitude (Δratio of F360/F380) of Ca 2+ response was of 0.55 ± 0.06 a.u. After a short latency, nearly 50% of surrounding MCs (a number of 50 MCs, out of 101) experimented [Ca 2+ ] i oscillations of Flores et al. Journal of Neuroinflammation (2019) 16:107 Page 3 of 13 variable pattern (Fig. 1b) and low amplitude (ΔF/F, 0.078 ± 0.009) (Fig. 1d). No calcium signal was observed when the depolarizing pulse was applied on a pure MC culture. Interestingly, after MC responses, a new but short [Ca 2+ ] i transient was observed in 16% of tested neurons (5 out of 31). This neuronal signal only occurred in neurons coculturedwithMCsbutnotinisolatedhippocampalneurons, and it could be reproduced for a second time when a new depolarizing pulse of high K + was again applied, suggesting that MC mediators feedback on neuronal activity. The amplitude of the second neuronal [Ca 2+ ] i peak was 0.039 ± 0.007. While MCs responses occurred 33.1 ± 2.9 s after the onset of first neuronal transient induced by depolarization, late Ca 2+ transients evoked in neurons after MCs response showed a similar delay of 30.4 ± 8.8 s (Fig. 1e). Activation of hippocampal neurons by mast cell degranulation The selective stimulation of MCs in close proximity to hippocampal neurons by compound 48/80 and antigen caused an immediate change in appearance of most MCs that led to degranulation within minutes. When MCs were activated with C48/80 (5 s application), a rapid Ca 2+ elevation Fig. 1 Calcium imaging of MC and Neuronal activity. aPhase-contrast image on the left panel of a hippocampal neuron (asterisk) at 2 weeks in culture following 24 h coculture with mast cells (black arrows). A glass micropipette of 1 μmϕwas positioned very close to the neuronal soma before pressure ejection (2.5 psi) pulses of 5 s. The right panel shows the same cells loaded with the Ca 2+ sensitive dye Fura-2 AM (MCs selected by dashed circles and neuron by the dotted polygon). Scale bar is 5 μm. bTraces of [Ca 2+ ] i transients in three mast cells (blue, red, and green traces) and a hippocampal neuronal cell body (black trace) triggered by the initial neuronal depolarization by microperfusion (70 mM [K + ] Locke solution). Note the [Ca 2+ ] i oscillation pattern elicited in MCs after initial neuronal activation and the short [Ca 2+ ] i transient (*) evoked in neuron later. Both responses were reproduced in a second stimulation. cPercentage of responding cells. dMean [Ca 2+ ] i peak obtained in MC- and neuron-evoked responses. eLatencies measured from the beginning of neuronal [Ca 2+ ] i transient induced by depolarization (a) to the origin of MC oscillations (b) and from the latter to the second rise in neuronal trace (c) (*) Flores et al. Journal of Neuroinflammation (2019) 16:107 Page 4 of 13 was elicited (ΔF/F, 0.21 ± 0.03) that persisted at mid-levels for minutes (Fig. 2a). The effect of MC degranulation by C48/80 on Ca 2+ signals of neighboring neurons occurred fairly rapidly (delay, 10.3 ± 2.9 s) (Fig. 2f) and consisted in an acute Ca 2+ transient with a peak amplitude of 0.24 ± 0.13 (Fig. 2e). The percentage of responding neurons was totaled 43%. MCs previously sensitized with monoclonal IgE against dinitrophenyl (DNP) and stimulated with the multivalent antigen DNP-conjugated human serum albumin (HSA) showed a mild degranulation response Fig. 2 MC to neuronal [Ca 2+ ] i signaling. aTraces of [Ca 2+ ] i transients in a mast cell (red trace) and a hippocampal neuronal cell body (black trace) in response to mast cell activation by C48/80 microperfusion (100 μg/ml for 30 s). Inset shows pictures of MCs morphology and appearance before and after stimulation. bTraces of [Ca 2+ ] i transients in a mast cell (red trace) and a hippocampal neuron (black trace) in response to mast cell activation by FcεRI receptor crosslinking by DNP microperfusion (1 μg/ml for 30 s). cTraces of [Ca 2+ ] i transients in a MC (red trace) and a hippocampal neuronal cell body (black trace) in response to MC activation by LPS (1 μg/ml for 1 min). dPercentage of responding cells to C48/ 80, DNP, and LPS. eMean [Ca 2+ ] i peak obtained in MCs (red bars) and hippocampal neurons (black bars) evoked by C48/80, DNP, and LPS. f Latencies measured by the time interval between the rise of MC [Ca 2+ ] i transient and the onset of neuronal [Ca 2+ ] i response Flores et al. Journal of Neuroinflammation (2019) 16:107 Page 5 of 13 evaluated by visual inspection of images using a bright-field microscopy. Antigenic stimulation (DNP) for 30 s elicited [Ca 2+ ] i oscillations in MCs (Fig. 2b) with a peak amplitude of 0.14 ± 0.01. Subsequently, 25% of hippocampal neurons responded after MC activation, since Ca 2+ waves of variable amplitude (ΔF/F,0.32±0.08)(Fig.2e) and delay (mean, 46.8 ± 22.6 s) (Fig. 2f) were observed. The application of LPS for 1 min to MCs only evoked a short burst of oscillations in [Ca 2+ ] i of low amplitude (ΔF/F, 0.06 ± 0.007) and took a long time to appear (Fig. 2c). MCs did not show morphological changes or apparent degranulation, and [Ca 2+ ] i signals from adjacent neurons remained unaltered. The possibility of direct effects of DNP or C48/80 on neuronal [Ca 2+ ] i signal was tested by applying these agents to hippocampal cultures in the absence of MCs. Neither DNP nor C48/80 alone had an effect on neuronal [Ca 2+ ] i signals. However, C48/80 has been said to exert a direct and strong excitatory action on enteric neurons and visceral afferents [30]. Therefore, in preventing potential direct effects of these agents on neurons, or indirectly, via mixed glia, we prepared a cocktail of secretory mediators from MCs (MCM). The cocktail of MC mediators (MCM) consisted of an extract obtained from degranulation by heat (53 °C, for 1 h) of 10 6 MCs per milliliter. Application of this MCM via a local pipette directly onto a hippocampal neuron cell body evoked a successful activation, measured by [Ca 2+ ] i transients, in 92% of cells (n=121) (Fig. 3a). Hippocampal neurons from a pure culture without glia were also able to react to the MCM similarly to neurons from mixed cultures. As a control, when the cocktail of mediators was obtained from MCs without degranulation (subjected to 37 °C instead of 53 °C, for 1 h), it failed to induce neuronal activation (n= 10). Therefore, hereinafter, we used the MCM obtained by heat (53 °C). In comparison with pure MCM, application of high K + -enriched solution evoked higher [Ca 2+ ] i transients with larger peaks (ΔF/F, 0.55 ± 0.06 vs 0.47 ± 0.03; p=0.021) (Fig. 3e) but a similar area under curve (or the integral over the deviation in [Ca 2+ ] throughout the transient) (2.62 ± 0.25 vs 2.7 ± 0.2 ) (Fig. 3f). Successive dilutions of this MCM concentrate (1:1; 1:3; 1:10) showed a dose response-dependence: [Ca 2+ ] i peak amplitude decreased from 0.36 ± 0.04, n=18 cells; (1:1 dilution) to 0.13 ± 0.03, n= 16 cells; (1:3 dilution) whereas the area under the curve decreased from 1.22 ± 0.15 (1:1 dilution) to 0.34 ± 0.08 (1:3 dilution). The last dilution of MCM (1:10) evoked a response in only 1 out of 9 tested neurons that showed a[Ca 2+ ] i peak amplitude of 0.8 and an area under the curve of 0.29. MC mediators elicit synaptic transmission in hippocampal neurons Transmitter release is tightly regulated by the presynaptic Ca 2+ transient [31,32]. Because MC degranulation is capable of evoking evident Ca 2+ transients in hippocampal neurons, we examined the transmitter of release using fluorescent imaging. Activity-dependent unloading of fluorescent styryl dyes (FM dyes) from recycling synaptic vesicles provides a direct measure of synaptic function at the level of single synapses [33]. Synaptic vesicles were loaded with FM1-43 dye for 60-s bath perfusion using 70 mM [K + ] solution. Subsequently, cells were continuously perfused for 5–10 min with 3 mM [K + ] basal solution in order to wash out excess FM dye from neuronal membranes. The cells showed a substantial amount of internalized probe (Fig. 4a, c) as a consequence of a first exo-endocytic process. Synaptic FM dye release was induced by applying 70 mM [K + ] solution (Fig. 4b) or MCM (Fig. 4d) for 5 s through a microperfusion pipette positioned at a distance of approximately 100 μm from the respective neuron. Destaining of synaptic FM dye clusters, defined by a monoexponential loss of fluorescence, was in both cases observed. The amplitude and the time course of synaptic FM dye release have been shown to provide direct readouts of the vesicle pool size and the release probability, respectively, at the level of single synapses [34]. Thus, in order to analyze release amplitudes (ΔF) and time constants (τ), FM dye release traces of individual synapses were fitted by a mono-exponential function (Fig. 4e, f). MCM was able to induce an evident FM-unloading indicating MC mediators can induce synaptic vesicle release and retrieval in cultured neurons. However, depolarization with 70 mM [K + ]solutioninduced a significantly higher (17.6 ± 1.4 a.u.; p=0.002;n=6 neurons) synaptic release amplitude than the MCM condition (8.7 ± 1.7 a.u; n=14 neurons) (Fig. 4g), suggesting the cocktail of MC mediators results in a weaker stimulus and recruits a smaller pool of vesicles than the depolarizing stimulus. In contrast, the rate of release (time constants,τ) was similar (high K + , 12.06 ± 0.42 s; MCM, 11.44 ±1.07s) (Fig. 4h) indicating MCM has the same release efficacy of the individual synapses as depolarization with 70 mM [K + ]solution. MC modulation of hippocampal neuronal function Next, we investigated which mediators were involved in the MC-neuron signaling. MCs contain an array of chemical mediators that can be released to the extracellular medium upon activation. We focused on the preformed products contained in the granules since the brief delay in neuronal reaction after MC activation (≈ 30 s) suggests a rapid release of mediators by exocytosis. To elucidate which mediators were involved in the promotion of neuronal activation, we first directly stimulated neurons with bioactive monoamines (histamine, serotonin) and ATP. All of them failed to evoke a [Ca 2+ ] i transient in hippocampal neurons. Neuronal [Ca 2+ ] i signal remained at baseline even for long applications (data Flores et al. Journal of Neuroinflammation (2019) 16:107 Page 6 of 13 not shown), suggesting these mediators by themselves are incapable of eliciting the rapid response observed by MCM. Second, we selectively blocked PAR2 receptors with the antagonist FSLLRY-NH2 to determine the involvement of mast cell-specific proteases. We incubated cells for at least 10 min with FSLLRY-NH2 (400 μM) before proceeding to apply MCM. However, the drug barely modified [Ca 2+ ] i signal evoked by MCM (ΔF, 106.19 ± 8.13%; area under curve, 75.87 ± 8.16%; n= 66)(Fig. 5b). Lastly, to resolve the implication of highly anionic serglycin proteoglycans (PGs) containing glycosaminoglycan side chains of either heparin or chondroitin sulfate (CS) type in neuronal activation, we incubated the MCM with heparinase or chondroitinase ABC for 1 h at 37 °C to break down the PGs of heparin and CS, respectively. Heparinase did not change the peak amplitude and area under curve of [Ca 2+ ] i transients respect to untreated MCM (ΔF, 100.09 ± 7.7%; area under the curve, 85.6 ± 10.3%; n= 57) (Fig. 5c). Conversely, chondroitinase ABC reduced the amount of [Ca 2+ ] i entry into the cell (Fig. 5d). Although [Ca 2+ ] i amplitude did not decrease significantly (ΔF, 88.81 ± 4.47%), [Ca 2+ ] i transient was narrower than that observed by untreated MCM, resulting in a lower area under the curve (63.3 ± 8.89%; p= 0.006; n= 57) (Fig. 5h), suggesting PGs may be in part responsible of MC-neuron communication. Fig. 3 Mast cell mediator cocktail (MCM) directly elicits [Ca 2+ ] i transients in hippocampal neurons. a[Ca 2+ ] i signals evoked by single pressure ejection pulses of 5 s of pure extract of MCM (solid trace) and 70 mM [K + ] Locke solution (dashed trace). The resulting calcium responses obtained by MCM was compared with calcium responses elicited by depolarization. bRepresentative [Ca 2+ ] i signal evoked by a single pressure ejection pulse of 5 s of MCM at 1:1 dilution. In order to estimate the minimal number of MCs necessary to obtain enough amount of mediators to induce a calcium transient in neurons, different dilutions from pure MCM were made. cRepresentative [Ca 2+ ] i signal evoked by a single pressure ejection pulse of 5 s of MCM at 1:3 dilution. d[Ca 2+ ] i signal evoked by a single pressure ejection pulse of 5 s of MCM a 1:10 dilution. e Mean [Ca 2+ ] i peak obtained in neuronal responses evoked by 70 mM [K + ] and MCM at 1:0 (pure), 1:1, 1:3, and 1:10 dilutions. fMean area under curve obtained from [Ca 2+ ] i transients in neurons evoked by 70 mM [K + ] and MCM at 1:0 (pure), 1:1, 1:3, and 1:10 dilutions. The latter was the lowest effective dilution capable of stimulating neurons. *p< 0.05, ***p< 0.001 Flores et al. Journal of Neuroinflammation (2019) 16:107 Page 7 of 13 Although, we combined both enzymes and increased incubation time of MCM to 24 h at 37 °C to make sure the complete degradation of PGs, we did not observe a stronger effect on [Ca 2+ ] i signal (data not shown). On the other hand, we decided to look into the mechanism that allows [Ca 2+ ] i to rise in neurons following MCM application. Accordingly, we examined the participation of glutamate receptors by using the NMDAR antagonist, D-2-amino 5-phosphonova- leric acid (D-APV), and the AMPA and kainate receptor blocker, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Cell Fig. 4 Results of FM imaging system in cultured hippocampal neurons. a,cSynaptic boutons of hippocampal neurons stained by FM1-43 dye in a loading process which consisted in 2 min of stimulation with 70 mM [K + ] and subsequent washing. The presence of numerous puncta of fluorescence that appear to be lined up along the neurites result from localized internalization of the dye and are not washed out. bSynaptic boutons destained after a stimulation pulse of 5 s with 70 mM [K + ]-enriched solution or dwith MCM. Note the marked decrease in fluorescence intensity of most of the puncta visible in aand c. Scale bar is 5 μm and applies to all frames eNormalized destaining curves of FM dye loaded synapses upon stimulation with high K + and fMCM. FM dye release curves of all single synapses were fitted mono-exponentially. gAmplitude (ΔF) and hτwere measured from monoexponential fitting curves obtained by depolarization and MCM, and plotted. Data represent average values of 6 (70 mM [K + ]) and 14 (MCM) neurons. **p< 0.01 Flores et al. Journal of Neuroinflammation (2019) 16:107 Page 8 of 13 incubation of D-APV 50 μM for 10 min at 37 °C before MCM perfusion achieved the most important reduction in [Ca 2+ ] i rise elicited by the cocktail of MC mediators (Fig. 5f) (ΔF, 61.15 ± 12.14%; p= 0.047; area under the curve, 35.54 ± 8.51%; p= 0.021; n= 25). However, the inhibition of AMPA and kainate receptors with CNQX 20 μM (after cell incubation for 10 min, at 37 °C) did not modify the [Ca 2+ ] i transient evoked by MCM (ΔF,96±9.9%;areaunderthe curve, 85.04 ± 11.7%; n= 28) (Fig. 5e). In sum, the results of pharmacological experiments indicate that MC granule mediators elicit calcium transients and exocytosis by activating NMDA receptors in hippocampal neurons and partly, at least, PGs of CS rather than heparin contribute to the mediation of these effects on neuronal physiology. Fig. 5 PGs of CS and NMDA-receptors contribute to outline the [Ca 2+ ] i signal elicited by mast cells mediator cocktail. aRepresentative time course of the elevation of [Ca 2+ ] i upon application of a brief pulse of MCM to a neuronal soma. bThe response was not changed after incubation with FSLLRY-NH2 and cHeparinase, but dwas decreased when MCM was pre-digested with enzyme chondroitinase ABC. e,fExamples of [Ca 2+ ] i signals elicited after CNQX 20 μMandD-APV50μMtreatment.gAveraged results on the magnitude of the peak of [Ca 2+ ] i and harea under curve are presented as percentage of means ± SEM. Chondroitinase inhibited partially the [Ca 2+ ] i signal as well as the NMDA receptor selective antagonist, D-APV, that blocked [Ca 2+ ] i response by 65%; however, the AMPA and kainate receptors blocker, CNQX, did not affect it. *p<0.05;**p<0.01 Flores et al. Journal of Neuroinflammation (2019) 16:107 Page 9 of 13