Aim: We hypothesize that both type-1 ryanodine receptor (RyR1) and IP3-receptor (IP3R) calcium channels are necessary for the mitochondrial Ca2+ increase caused by membrane depolarization induced by potassium  of single skeletal muscle fibers; this calcium increase would couple muscle fiber excitation to an increase in metabolic output from mitochondria (excitation-metabolism coupling).Methods: Mitochondria matrix and cytoplasmic Ca2+ levels were evaluated in fibers isolated from flexor digitorium brevis muscle using plasmids for the expression of a mitochondrial Ca2+ sensor (CEPIA3mt) or a cytoplasmic Ca2+ sensor (RCaMP). The role of intracellular Ca2+ channels was evaluated using both specific pharmacological inhibitors (xestospongin B for IP3R and Dantrolene for RyR1) and a genetic approach (shIP3R1-RFP). O2 consumption was detected using Seahorse Extracellular Flux Analyzer.Results: In isolated muscle fibers cell membrane depolarization increased both cytoplasmic and mitochondrial Ca2+ levels. Mitochondrial Ca2+ uptake required functional inositol IP3R and RyR1 channels. Inhibition of either channel decreased basal O2 consumption rate but only RyR1 inhibition decreased ATP-linked O2 consumption. Cell membrane depolarization-induced Ca2+ signals in sub-sarcolemmal mitochondria were accompanied by a reduction in mitochondrial membrane potential; Ca2+ signals propagated toward intermyofibrillar mitochondria, which displayed increased membrane potential. These results are compatible with slow, Ca2+-dependent propagation of mitochondrial membrane potential from the surface toward the center of the fiber.Conclusion: Ca2+-dependent changes in mitochondrial membrane potential have different kinetics in the surface vs. the center of the fiber; these differences are likely to play a critical role in the control of mitochondrial metabolism, both at rest and after membrane depolarization as part of an “excitation-metabolism” coupling process in skeletal muscle fibers. The skeletal muscle system plays a critical role in the energy balance of the organism. During contraction, its energy requirements can increase nearly instantaneously by more than 100-fold  from the matrix to the intermembrane space (IMS). This process contributes to create a large mitochondrial membrane potential (ΔΨmt) that, besides its key role in ATP synthesis, produces a large driving force for Ca2+ uptake into the mitochondrial matrix  complex, recently identified and characterized  process, represents a fast [Ca2+] rise that emerges from Ca2+ release through ryanodine receptor type-1 (RyR1) Ca2+ channels. The second Ca2+ transient is slower and depends on activation of the inositol 1,4,5-trisphosphate receptor (IP3R); this slow transient relates to gene expression and mediates the excitation-transcription coupling (ETC) process  is amenable to transfer to the mitochondria has been proposed in other cell types  CEPIA3mt (Ca2+-measuring protein indicator type 3). We electroporated adult FDB muscles in vivo with a plasmid that promotes the expression of the CEPIA3mt sensor protein with selective mitochondrial destination  after stimulation; the addition of 0.5 μM FCCP (Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) caused a similar dissipation of this increase in both selected ROIs  and CEPIA3mt and subjected the isolated fibers to electrical stimulation (ES). As shown in Figure 2+ signals. Exposure to the high K+ solution induced a similar increase in RCamPs and Cepia3mt fluorescence  was determined after the projection of the standard deviation of fluorescence over time Figure . Membranr Figure . The maxr Figure . To ensue Figure ; likewiss Figure . The timd Figure , suggest+ during 1 min induced muscle fiber damage, we performed a control on the excitability of the fiber. To this aim, muscle fibers loaded with Fluo4-AM were exposed to electrical stimulation (ES) or high K+ medium. As shown in Figure + medium induced a smaller and longer (≈10 s) calcium transient compared to ES. 40 s after removal of the high K+ medium, the muscle fibers recovered their response to ES suggesting that treatment with high K+ (65 mM during 1 min) did not affect the long term viability of the muscle fibers  or xestospongin B (10 μM). The maximal fluorescence observed was 66.2% (±8.0) in the control, 29.5% (±1.3) in dantrolene pre-treated fibers and 33.0% (±1.9) in xestospongin B pre-treated cells . These results are consistent with the kinetics of activation of RyR1 (fast) and IP3R (slow) in muscle fibers. Furthermore, these findings suggest that Ca2+ release through both IP3R and RyR1 are involved in the mitochondrial Ca2+ increase produced by depolarizing stimuli.The two main intracellular Cas Figure . Furthery Figure ,  increase in mitochondrial Ca2+ levels after each photo-release (10 flashes) of IP3 compared with the control .In order to corroborate the contribution of IPy Figure . In line Figures . Additio Figures . The amp3R1- and RyR1-mediated Ca2+ release contribute to the mitochondrial Ca2+ increase, and thus to the mitochondrial functional activation triggered by muscle stimulation (and contractile activity).Altogether, these findings indicate that activation of both IP3R and RyR1 are necessary to induce the metabolic increase caused by membrane depolarization. To test this hypothesis, OCR was evaluated after depolarization in muscle fibers pre-incubated with the specific inhibitors xestospongin B and dantrolene. We first focused on the role of IP3R on membrane depolarization-dependent mitochondrial OCR increase. As previously shown, depolarization with high K+ increased both, the basal and the ATP-linked OCR  in a non-quenching mode. As shown in Figure 2+ levels evaluated by CEPIA3mt; ΔΨmt reduction exhibited a delay (10–15 s) compared to the mitochondrial Ca2+ increase . According to the results described above, ES induced a similar effect on Ca2+ handling as did K+-induced membrane depolarization on ΔΨmt. ES reduced ΔΨmt in the SSM region with a maximal effect at 35s (±4 s), and this reduction was reversed after 10 min   or downregulation (shIP3R-RFP), and that locally photo-released IP3 increased mitochondrial Ca2+ levels in 50% of the cells evaluated. Based on these combined results we strongly suggest that both RyR1 and IP3R mediate the mitochondria Ca2+ increase detected after depolarization of adult skeletal muscle fibers.Although a mitochondrial calcium increase after depolarization has been already studied in isolated adult muscle fibers using electrical stimulation or high potassium medium  because of the high concentration reached by this ion in the cytosol, the direct transference of Ca2+ from SER to the mitochondria through the IP3R channel might explain why both RyR1 and IP3R channels contribute in a similar magnitude to the increase of mitochondrial Ca2+ level after depolarization. Furthermore, the two mitochondria populations appear to have a differential content of proteins (MICU1) needed for calcium entry regulation.We can speculate then that about half of the mitochondria in this particular muscle fiber will be located in the subsarcolemmal region, in close contact with SER that contains IP2+ sensor Cepia3mt  exhibited significant preferential mitochondria localization . It was previously reported that in muscle fibers the maximal mitochondrial Ca2+ increase reached during a prolonged depolarization pulse exhibits a small delay compared to the cytoplasmic Ca2+ transient  in HEK-293 cells reported that RyR1-MH enhanced mitochondrial Ca2+ levels  , thus the ΔΨmt may be distributed toward IMFM to generate ATP in the intermyofibrillar region  increase. In fact, the calcium increase in the SSM is larger and frequently precedes the calcium changes in IMFM, possibly due to differential mitochondrial protein distribution; this may be influencing the effect of Ca2+ over ΔΨmt in these two regions.The SSM in their location, function and spatial organization are quite different to the IMFM  muscle was obtained from C57BL6/J mice between 8 and 10 weeks of age as previously described . The ES protocol was a single sequence of 270 square pulses of 0.3 ms duration at 20 Hz or 90 Hz (lasting 13.5 or 3 s) with platinum electrodes wires intercalated 0.5 cm apart, as described previously .The following plasmids were used. pCMV-Cepia3mt (Invitrogen N° 58219), pCMV- RCamP 1.07 (RCamPs)  Seahorse medium or control isotonic Seahorse medium during 1 min. Then, each well was washed with control seahorse medium and the plate was incubated for 5 min. At the end of this time, OCR was evaluated. To analyze mitochondrial function, fibers were treated sequentially with oligomycin (10 mg mL−1), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone , and rotenone (1 μM). OCR was recorded following each injection. Each experimental condition was assigned to 16 wells per plate . The OCR value of each well was normalized against its own protein concentration. We excluded from the analysis the wells that contained less than 1 μg of protein, corresponding to 30% of the total wells evaluated by each condition. In order to avoid the contamination with myoblasts, the purity of the muscle fibers culture was over 95%.Oxygen Consumption Rate (OCR) was evaluated using XF96 Seahorse Extracellular Flux Analyzer (Seahorse Agilent Technologies ©). Fibers obtained from 4 mice were plated 12 h before the experiment, in Seahorse XF+ 0.206 μg/μL (±0.04); K+ + Xesto. B 0.186 μg/μL (±0.04); K+ + Dantro. 0.119 μg/μL (±0.02). The protein concentration of each well was determined immediately after de OCR assay.The protein concentration in each condition was: control 0.135 μg/μL (±0.02); Xesto. B 0.158 μg/μL (±0.02); Dantro. 0.16 μg/μL (±0.02); K2, 1 MgCl2, 5.6 glucose, 10 HEPES, pH 7.4 at room temperature in the presence of 75 μM N-benzyl-P-toluenesulfonamide  to inhibit muscle contraction. The real-time imaging experiments were performed by confocal microscopy using Carl Zeiss Pascal 5, LSM with a PlanApo 64 × /1.4 N.A. oil immersion objective, Leica TCS-SP5-II with a 100 × /1.4 N.A. Plan-apochromat objective equipped with STED system. The pixel size was set below 100 nm to meet the Nyquist criterion. Pinhole size was set at 1 Airy unit and z-stacks were acquired with a step size of 130 nm. Lateral and axial resolutions were approximately 230 and 460 nm, respectively; determinations were done in an inverted Olympus IX81 microscope with a 40 × /N.A. 1.3 oil immersion objective or in a spinning disk microscope  Plan-NEOFLUAR 100 × /1.3 N.A. oil immersion objective. Muscle depolarization was performed using the isotonic high K+ medium (65 mM) or ES.All experiments were conducted in Krebs Ringer buffer (in mM): 145 NaCl, 5 KCl, 1 CaCl2+ measurement was performed 7 days after in vivo electroporation with plasmids for expression of either Cepia3mt or mtGcaMP6m. The electroporation was performed according to previously reported procedures *100. Mitochondrial Ca2+ level at resting condition was evaluated by measuring mtGcaMP6m fluorescence in the inverted Olympus IX81 microscope. Seven days after electroporation muscle fibers were isolated and mtGcaMP6 fluorescence was detected using an excitation/emission wavelength λexc1–λexc2/λem = 400–490/520 nm. The ratio between the signals excited with 490 and 400 nm was used to determine the Ca2+ level. Noise in the images was corrected frame by frame by subtracting the mean pixel value of a cell-free region of interest (ROI) using Fiji distribution of ImageJ ; for analysis 20 ROI were selected per fiber.Mitochondrial Cain vivo electroporation with the cytosolic Ca2+ indicator red GECI (RCamPs) *100. The background was corrected against the mean pixel value of a cell-free ROI using Fiji. During experiments, the fibers were maintained in Krebs buffer and images were acquired every 1 s. For the analysis 20 ROI were selected per cell.Muscle fibers were isolated 7 days after +, Life Technologies) for 40 min at 37°C plus mitotracker green (MTG). MTG was used to normalize the fluorescence among the different mitochondrial populations. TMRE+ fluorescence was detected using the excitation-emission λ545–580/590 nm using Carl Zeiss Pascal 5 microscope. For kinetic determinations, images were acquired every 1 s with 1% laser gain maintained under 1%, in order to avoid quenching and fluorescence toxicities. FCCP (0.5 μM) or Oligomycin (1 μM) was added at the end of the experiment to blunt-collapse ΔΨmt. The ΔΨmt was evaluated as raw fluorescence intensity of background-corrected images by subtracting the fluorescence intensity mean value after FCCP or oligomycin administration from the average value of the first 10 acquisitions (ΔF).ΔΨmt was measured by loading fibers with 20 nM tetramethylrhodamine, ethyl ester benzyl-myo-inositol-triphosphate-hexakis(propionoxymethyl) ester; Sichem GmbH) during 45 min at 37°C. Fibers were placed in the microscope under the beam of the UV laser . After acquiring a 20 s baseline at 10 Hz, photorelease of caged IP3 was performed by applying a 435 nm UV laser at 15 Hz to a mitochondrial local spot (~3–4 μm in diameter). Fluorescence signals were recorded at 10 Hz in a spinning disk microscope . The results were processed with the formula (ΔF/F0)*100. The background signal recorded in cell-free regions was used to correct the fluorescence.Muscle fibers were electroporated with a plasmid for expression of Cepia34Cl in PBS for 10 min. Cells were washed with ice-cold PBS and permeabilized for 20 min with 1% Triton X-100 in PBS and blocked in PBS-BSA 4% (w/v) during 45 min. Fibers were incubated in PBS-BSA 2% (w/v) with antibody against TOM20  1:100; Cytochrome C  1:100, complex IV  1:100 and MCU  1:50, MICU1  1:50 and anti-subunit alpha of the ATPase  1:50 overnight at 4°C. Finally, cells were washed three times with PBS during 5 min each and incubated with secondary antibody anti-mouse and anti-rabbit Alexa Fluor 488/Alexa Fluor 546 as appropriate and coverslips were mounted with ProLong Gold Antifade reagent (Life Technologies). Images were taken in both x,y and x,z-axis scan using the Leica TCS-SP5-II microscope equipped with 40x, 1.25 N.A., Plan-apochromat objective, in STED configuration. 488 nm and 543 nm laser lines and images were collected in the 496–550 nm and 580–690 nm ranges. The pinhole was set to 1.0 airy units and pixel size was set to 48.88 nm. Image deconvolution and processing were performed using Fiji distribution of ImageJ. Mander's coefficients were calculated using the JACoP plugin for Fiji  in a lysis buffer containing in mM: 20 Tris-HCl (pH 7.5), 1% Triton X-100, 2 EDTA, 20 NaF, 1 Na2P2O7, 10% glycerol, 150 NaCl, 10 Na3VO4, 1 PMSF and protease inhibitors . Protein separation was performed using SDS-PAGE followed by transfer to PVDF membranes. The following primary antibodies were used: anti-IP3R1 and horseradish peroxidase-conjugated secondary antibodies . The protein bands in the blots were visualized using a WESTAR Supernova detection kit  and ChemiDoc™ MP System . The intensity of the bands was determined by ImageJ densitometry analysis.One week after electroporation with shIPt-test and one-way ANOVA followed by Dunnett's post-test respectively. For multiple paired comparisons, One-way ANOVA followed by Bonferroni's post-test was performed. P < 0.05 was considered statistically significant. Graph-Pad PRISM version 5.0 software  was used for data fitting.All experimental results are expressed as means ± S.D. For paired data and for multiple comparisons we used Student's AD-V designed and performed the majority of the experiments; he wrote and discussed the manuscript. AC performed and analyzed the photo-release of IP3 experiments. DV discussed and wrote part of the manuscript; she participated in the data analysis. PL performed knock-down of IP3R experiments. CH contributed in data discussion, writing and discussion of the manuscript. GG performed and analyzed MEF-cells KO for MICU1; she collaborated in immunofluorescence measurements of intramitochondrial proteins. DD participated in data discussion, writing and discussion of the manuscript. CM participated in data discussion, writing and discussion of the manuscript. RR participated in data discussion, writing and discussion of the manuscript. AC-F participated in the main idea of the manuscript, design of experiments, data analysis and discussion of the manuscript. EJ participated in planning, design of experiments and the main idea of the manuscript, data discussion and manuscript writing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The CCh-induced increase in calcium concentration was significantly lower in the presence of extracellular Ca2+ in the long-term dexamethasone treatment group compared to that in the control group. Furthermore, CCh-induced salivation in the absence of extracellular Ca2+ and Ca2+ ionophore A23187-induced salivation was comparable between the control and long-term dexamethasone treatment groups. Moreover, salivation induced by the Ca2+-ATPase inhibitor thapsigargin was diminished in the long-term dexamethasone treatment group. In summary, these results demonstrate that short-term dexamethasone treatment did not impair salivary gland function, whereas long-term dexamethasone treatment diminished store-operated Ca2+ entry, resulting in hyposalivation in mouse submandibular glands.Corticosteroids are used in the treatment of many diseases; however, they also induce various side effects. Dexamethasone is one of the most potent corticosteroids, and it has been reported to induce the side effect of impaired salivary gland function. This study aimed to evaluate the effects of dexamethasone on mouse submandibular gland function to gain insight into the mechanism of dexamethasone-induced salivary hypofunction. The muscarinic agonist carbachol (CCh) induced salivary secretion and was not affected by short-term dexamethasone treatment but was decreased following long-term dexamethasone administration. The expression levels of the membrane proteins Na Long-term corticosteroid use causes dry mouth by inhibiting calcium movement into salivary gland cells. Corticosteroid use is associated with multiple systemic side effects including salivary inhibition, prompting Yusuke Kondo and a research team from Japan’s Kyushu Dental University to elucidate the reasons why. Submandibular glands taken from mice after six weeks’ treatment with the corticosteroid dexamethasone produced significantly less saliva in ten minutes than a group given just one week’s treatment, or no treatment at all. Further investigation revealed that long-term dexamethasone treatment inhibited the ability of salivary gland cells, known as acinar cells, to transport calcium ions from the exterior to the interior of the cells—an essential mechanism enabling salivation. The study improves our understanding of corticosteroid-induced dry mouth and reveals potential pharmacological targets to enable treatment. Dexamethasone, a potent glucocorticoid, can cause salivary alterations,11 The salivary glands are composed of acinar cells and ductal cells, and many channels and transporters contribute to salivary secretion.13 Activated muscarinic receptors lead to G protein-regulated activation of phosphatidylinositol 4,5, bisphosphate (PIP2)-specific phospholipase C and PIP2 hydrolysis, which results in the generation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. IP3 activates the IP3 receptor, which is expressed on the endoplasmic reticulum (ER) membrane. Activated IP3 receptor induces Ca2+ release from the ER, which results not only in an increase in intracellular calcium concentrations ([Ca2+]i) but also in depletion of Ca2+ in the ER. Depleted ER Ca2+ causes extracellular Ca2+ entry, which is referred to as store-operated Ca2+ entry (SOCE).15 An increase in [Ca2+]i is essential for salivary fluid secretion, and impaired [Ca2+]i increase is associated with salivary hypofunction in patients with Sjögren’s Syndrome.16 Increased [Ca2+]i induces Cl− movement through the transmembrane protein 16A (TMEM16A) Ca2+-activated Cl− channel.19 The accumulation of Cl− in the lumen induces water movement through the aquaporin 5 (AQP5) water channel and tight junctions. The Na+-K+-2Cl− cotransporter (NKCC1) and anion exchanger move Cl− into acinar cells, consequently maintaining continuous salivary secretion.21 NKCC1 also plays a substantial role in determining the amplitude of oscillatory Cl− currents in salivary acinar cells.22 Furthermore, the ductal epithelial Na+ channel and cystic fibrosis transmembrane conductance regulator aid in Na+ and Cl− reabsorption from saliva, respectively.Dry mouth is caused by salivary gland dysfunction and results in oral mucosal pain, dysphagia, stomatitis, difficulty wearing dentures, and increased risk of dental caries and periodontal disease.23 Moreover, NKCC1 expression was reduced by glucocorticoids in alveolar and bronchial cells.24 Furthermore, dexamethasone treatment decreased [Ca2+]i in dendritic cells,25 pancreatic β-cells,26 and bronchial epithelial cells.27 Another study reported that dexamethasone decreased [Ca2+]i and consequently inhibited Cl− secretion in human bronchial epithelial cells.28 Therefore, we hypothesized that dexamethasone may directly affect salivary acinar cell function and that the expression of channels and transporters in submandibular acinar cells  or intracellular Ca2+ signalling may be impaired by dexamethasone treatment, which is true for other cells and tissues,30 resulting in dry mouth. However, the effects of dexamethasone on channels, transporters, and intracellular Ca2+ signalling in salivary glands are currently unknown.Previous reports have demonstrated that glucocorticoids affect the expression or function of channels and transporters in some tissues. For example, dexamethasone increased AQP1 expression and water transport in rat peritoneum.As shown in Figs. 2+ ionophore, induced salivation in ex vivo SMGs. The CCh-induced salivary flow rate is shown in Figs. 2+]i increase in Fura 2-AM-loaded SMG acinar cells  were purchased from CLEA Japan . Mice were maintained under a 12-h light/dark cycle and fed ad libitum. All experiments were approved by the Animal Committee of Kyushu Dental University (No. 16–011). Collagenase L was purchased from Nitta Gelatin ; Fura 2-AM was purchased from Dojindo Molecular Technologies, Inc. ; 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) was purchased from Life Technologies Corporation ; T16Ainh-A01 was obtained from Tocris Bioscience ; the NKCC1 antibody (sc-21545) was obtained from Santa Cruz Biotechnology ; the TMEM16A antibody (ab53213) was purchased from Abcam ; the AQP5 antibody (sc-9890) was purchased from EMD Millipore ; and the secondary antibody (Histofine Simple Stain Mouse MAX peroxidase) was purchased from Nichirei . All other reagents were purchased from Sigma-Aldrich Japan .−1.Ex vivo perfusion of the submandibular gland (SMG), ion imaging in dispersed SMG acinar cells and histological analysis studies were performed using a comparative study design. We evaluated the expression of three membrane proteins, TMEM16A, AQP5, and NKCC1, using immunohistochemistry. In the experimental group, dexamethasone was administered via drinking water for 1 week (short-term treatment: Dex1) or 6 weeks (long-term treatment: Dex6), while the control groups received distilled water. The Dex1 and Dex6 groups were composed of different animals. The control groups that were established for the Dex1 and Dex6 groups were also composed of different animals. The Dex1 and Dex6 treatment groups were compared with their respective control groups. In a preliminary experiment, the amount of water intake per day was measured to be approximately 4.7 mL per day. We converted the maximum human dose for articular rheumatism, 8 mg perday, to the corresponding dose in mice based on body surface area. We thus determined the dexamethasone concentration to be 8 mg·L−1): HCO3− and Ca2+-containing solution (B+): 4.3 KCl, 120 NaCl, 25 NaHCO3, 5 glucose, 10 HEPES, 1 CaCl2, and 1 MgCl2, equilibrated with 95% O2 and 5% CO2; HCO3--containing and Ca2+-free solution: 4.3 KCl, 122 NaCl, 25 NaHCO3, 5 glucose, 10 HEPES, and 1 MgCl2, equilibrated with 95% O2 and 5% CO2; HCO3−-free and high Cl−-containing solution (B-): 4.3 KCl, 120 NaCl, 25 Na gluconate, 5 glucose, 10 HEPES, and 1 MgCl2, equilibrated with 100% O2; and HCO3−-free and low Cl− solution: 4.3 K gluconate, 122 Na gluconate, 25 NaHCO3, 5 glucose, 10 HEPES, and 1 MgCl2, equilibrated with 100% O2.Solutions were as follows .39 In brief, mice were anaesthetized with chloral hydrate , and the maxillary artery, superficial temporal artery, lingual artery, thyrocervical artery, and internal carotid artery were ligated. Next, the common carotid artery was ligated and cut at the proximal portion of the internal and external carotid branch, and the SMG was removed along with the common carotid artery and duct. The carotid artery was immediately cannulated and perfused with physiological saline solution (B+as described above) at a flow rate of 1 mL·min−1 at 37 °C. The perfused SMG was stimulated with a muscarinic agonist , calcium ionophore , or Ca2+-ATPase inhibitor , and the secreted saliva was collected into a glass capillary tube.Perfusion of the SMG was performed as reported previously.40 Briefly, 5-µm thick sections were deparaffinized and rehydrated, and the antigens were unmasked with pH 6.0 citrate buffer solution at 95 °C for 20 min. Endogenous peroxidase activity was inhibited using 0.1% H2O2. The sections were then incubated for 60 min with an anti-peptide antibody against TMEM16A (1:1), AQP5 (1:200), or NKCC1 (1:1 000), followed by a secondary antibody at room temperature for 30 min to detect bound immunoglobulins, which were visualized using 3,3‵-diaminobenzidine solution at room temperature for 5 min. The sections were then stained with haematoxylin for 1.5 min. Finally, the sections were dehydrated in ethanol and mounted.For histological analysis, the excised SMGs were fixed in 4% paraformaldehyde phosphate buffer solution for 48 h before being embedded in paraffin. The sections (5-µm thick) were deparaffinized and rehydrated and stained with haematoxylin and eosin (HE). Immunohistochemistry for NKCC1, TMEM16A, and AQP5 was performed as previously described.−1, i.p.). Acinar cells were isolated by collagenase treatment with 520 U·mL−1 collagenase L for 15 min at 37 °C. The isolated cells were incubated with 2 µmol·L−1 Fura 2-AM for 10 min at 37 °C. Fluorescence was detected under a microscope equipped with a fluorescence analysis system  at an excitation wavelength of 340 or 380 nm and an emission capture of 510 nm. The [Ca2+]i was determined as the fluorescence ratio at 340/380 nm. The degree of [Ca2+]i increase during the stimulation periods was calculated from the integral value of [Ca2+]i as the area under the curve.SMGs were removed from the mice after they were anaesthetized with chloral hydrate  at an excitation wavelength of 340 nm and an emission capture of 510 nm. NKCC1 activity was examined in SPQ-loaded SMG acinar cells as previously described.40The isolated cells were incubated with 5 mmol·L− removal induced Cl− efflux, and extracellular Cl− restoration induced Cl− influx into the acinar cells. Note that the Cl− influx rate represents the NKCC1 activity in the presence of T16Ainh-A01, a TMEM16A inhibitor, and 6-ethoxy-2-benzothiazolesulfonamide (EZA), a carbonic anhydrase inhibitor, and in the absence of HCO3- because NKCC1 is the exclusive pathway for Cl− influx under this condition.In brief, extracellular Clt-test. Multiple-sample comparisons were performed using one-way ANOVA followed by Bonferroni’s post hoc test. P < 0.05 was considered statistically significant. The results are expressed as the mean ± standard error of the mean(SE). All experiments were performed using at least three different mice for each condition, with n referring to the number of experiments performed.Differences between the control and experimental groups were evaluated using the unpaired Student’s


While the expression of CaBPs by photoreceptors, retinal interneurons and the output ganglion cells in the mammalian retina has been extensively studied, a general description is still missing due to the differences between species, developmental expression patterns and study-to-study discrepancies. Furthermore, CaBPs are occasionally located in a compartment-specific manner and two or more CaBPs can be expressed by the same neuron, thereby sharing the labor of Ca2+ buffering in the intracellular milieu. This article reviews this topic by providing a framework on CaBP functional expression by neurons of the mammalian retina with an emphasis on human and mouse retinas and the three most abundant and extensively studied buffer proteins: parvalbumin, calretinin and calbindin.Ca As a second messenger, Ca2+ has a crucial role in intercellular communication and other neuronal functions. Therefore, intracellular Ca2+ levels must be maintained in a narrow and cell type-specific range. Besides the extrusion of Ca2+ into the extracellular space and sequestering in intracellular stores, living cells (including neurons) also utilize a set of Ca2+-binding buffer proteins (CaBPs) to adjust their actual Ca2+ concentration levels to the momentary functional requirement  and proteins of the S100 family are used as neuronal markers in the retina [Akin to other CNS loci, CaBPs have been shown to be widely expressed by neurons of the mammalian retina as well. In fact, there have been numerous approaches to reveal the identity of the diverse CaBP-expressing neuronal subtypes in retinas of various mammalian model species and the human tissue as well. CaBPs are divided into well-conserved protein families: annexins, pentraxins, C2-domain proteins, EF-hand proteins, vitamin-K-dependent proteins and the intraorganellar low-affinity, high-capacity Caproteins . HoweverThe mammalian retina is a well-organized, layered structure that serves the first steps of visual perception, including the phototransduction as well as initial analysis of information and transmits this towards the brain. It is divided into ten layers with five neuron classes including retinal ganglion cells (rGCs), amacrine cells (ACs), bipolar cells (BCs), horizontal cells (HCs) and photoreceptors (PRs) . These cDrosophila [Drosophila counterparts [Out of all the CaBPs, CaM is the most ubiquitous, found in all eukaryotic organisms including yeasts. It has a central role in regulating cell function and protein expression including CaMKII, which has been described (among many other functions) as an important regulator of gap junction conductance ,4. The eosophila , it coulterparts .2+-binding sites and two, further modified, domains lost their Ca2+-binding capability [2+ inflow induced by glutamate receptor stimulation and in Huntington disease [2+ buffering [2+ buffer widely expressed in the retina and other tissues as well, with two functional Ca2+-binding sites [2+ sensor in cone photoreceptors (but not in rods) of the murine retina at the PR-BC synapses [CaB is a 28-kDa globular-shaped protein that contains six EF-hand (helix-loop-helix motif) domains. Four of them form the two active Capability . Importa disease ,10. CaR, disease . Five ousynapses ,13. REC synapses . Even th2+ contain negatively charged groups arranged into a specific geometry that enhance chemical coordination of Ca2+. Proteins with appropriately spaced acidic side-chain residues  and/or backbone carbonyl groups provide the ‘cage’ in which a Ca2+ fits in. Different types of CaBPs  [2+. In general, the term ‘Ca2+ buffer’ is applied mostly to a small subset of cytosolic proteins of the EF-hand family, including PV and isoforms, CaB-D9k (9 kDa), CaB-D28k (28 kDa) and CaR. Some EF-hand proteins, such as CaM and S100, are ‘Ca2+ sensors’ that upon Ca2+ binding, change conformation and interact with specific targets [2+ sensors also function as Ca2+ buffers.The molecules serving as chelators for Caroteins) ,15 diffe2+ affinities and kinetics (binding and dissociation speeds) of CaBPs, which in turn modulate the spatiotemporal aspects of cytosolic Ca2+ signals and spiking activity of neurons. For example, due to the delayed Ca2+ binding of PV, the rising phase of Ca2+ concentration increase does not change (first 100 ms). On the other hand, the initial phase of the Ca2+ concentration decay is enhanced as PV finally takes effect, thus the intracellular Ca2+ wave becomes more transient. In addition, during the later decay phase of a signal, Ca2+ start dissociating from PV, enhancing neuronal activity. At the same time, CaB and CaR bind Ca2+ faster (even during the Ca2+ concentration rising phase), thus truncating stimulus-elicited Ca2+ responses (and spiking frequency) throughout the depolarization [2+ buffering by CaB also delays the Ca2+-dependent inactivation of voltage-dependent Ca2+ currents [Members of the EF-hand family show a structural diversity in their EF-hand motifs . These sge first 00 ms. Onge first 00 ms. Oncurrents .In summary, the actions of various CaBPs greatly affect neuronal excitability, activity patterns ,19 and f2+ concentration drops and inhibition by REC is decreased. In turn, rhodopsin kinase is released from inhibition and can phosphorylate metarhodopsin II (activated form of rhodopsin) that leads to a more rapid inactivation and faster kinetics [2+-bound form of REC in dark slows down the activity of rhodopsin kinase, thereby prolonging the light sensitivity of rhodopsin. Interestingly, REC is located in the cytosol when the intracellular Ca2+ level is low, and the ion is not bound to the protein. On the other hand, upon Ca2+ binding, a conformation change reveals the hidden myristoyl group that can now anchor to the disc membrane [REC is the only neuronal CaBP that is expressed in all PR cells in all examined mammalian species , includikinetics . The Ca2membrane .Besides REC, other CaBPs seem to be expressed more selectively. Cones have been successfully detected with antibodies that recognize CaB in various mammalian species, including humans and other primates ,42,43,44REC HCs receive their input from PRs and provide lateral feed-back inhibition to PRs and/or feed-forward inhibition to BCs ,48,49. THCs in most mammalian species express CaBPs ,56,66,67There have been 11–14 morphologically distinct BC subtypes identified in a wide range of mammalian retinas, including the mouse and the human retinas ,35,44,65Microcebus murinus), which is also a nocturnal animal [2+-dependent mechanisms of expressing BCs such as neurotransmitter release. Alternatively, REC may regulate the activity of kinases in a similar fashion as REC inhibits rhodopsin kinase in rods.At least four out of these eight human cone BC populations can be stained with sera recognizing CaBPs. REC is unique amongst CaBPs as it is specifically expressed (besides PRs) by FMB cells ,34,35. Wl animal . WhetherContrary to REC, CaB is expressed in at least three cone BC subpopulations in the human retina: in one OFF cell ; CaR and PV, similar to CaB, are not expressed by murine BCs and are largely absent from BCs in retinas of the other mammalian species as well. There is one description on CaR/CaB dual expression in one set of human BCs that shows morphological similarities with IMB or DB4 cells ,35,44. T2+ transient rising phases to serve as a Ca2+ source for later phases, decreasing the effects of the Ca2+-dependent inactivation of voltage-dependent Ca2+ currents, thereby prolonging the response decay [As discussed above  CaB is cse decay ,84,85,862+ buffer family, Haeseleer and colleagues [2+-binding buffer proteins with high homology to calmodulin  that are expressed in the mammalian retina. CaBP1 and CaBP5 has been shown in different subsets of BCs. CaB5-expressing cells in the human and macaque retinas have been identified as OFF DB3 and ON DB4 BCs, whereas CaBP+ cells in the mouse showed morphological similarities to the type3 (OFF) and type5 (ON) BCs [Besides members of the classical EF-hand Calleagues  identifi(ON) BCs . Finally(ON) BCs . HoweverThere is also insufficient information about the signaling pathways that are initiated in the retina by the various CaBP-expressing BCs, the DB6 BC being the only documented exception . The CaBAs the inhibitory neurons of the inner retina, ACs contribute to vision by modulating the activity of inner retinal pathways through a diverse network of synaptic connections. As a consequence, ACs display a rich variety in terms of both their function and morphology, and 30–40 different types have already been identified till the present day ,91,92. AIt is challenging to compare the above description on CaBP expression in the mouse retinal ACs  with those reported in humans  due to t2+ imaging and the matching of morphological and functional populations has been a subject of ongoing research. The light-evoked RGC responses could be ON, OFF or ON/OFF based on their polarity, but there are great differences in response kinetics that could also serve as a basis for classification.RGCs in the primate, monkey, cat, rabbit, and mouse retinas include a number of various morphological subtypes . The claLike the model species, over 20 different GC subtypes of the human retina have been characterized based on morphological criteria by Kolb and colleagues . M magn. HoweverThe CaBP expression patterns of RGCs in the mouse retina, as one of the most popular models, appear somewhat similar to the human retina . One of Following dye injections into CaR-immunolabeled mouse RGCs, Lee and colleagues identified 10 subtypes , whose Further experiments are required to prove whether the human counterparts of the CaR-, PV- and CaB-expressing mouse RGCs exist, and whether their CaBP signature is similar to those exhibited by mouse RGCs. Until then, it remains uncertain whether the mouse retina serves a good model for human RGC studies.2+ imbalance. Due to the changing PV expression levels in several neurodegenerative conditions, it has been suggested that PV also plays a role in determining cell survival [In the previous sections, we provided an extensive overview on cell types of the mammalian retina (with focus on the mouse and human retinas) regarding the expression of the three discussed CaBPs, which have generally been utilized as cell type-specific markers. However, it has been shown that CaBP expression can change under certain neurodegenerative conditions. For example, a reduced PV and or CaR expression was reported in cells of the INL and GCL as a result of diabetic retinopathy, retinal ischemia and glaucoma ,141,142)survival ,147,148.survival . Besidessurvival ,151,152.survival  and CaB+survival . It has survival ,155. Allsurvival  as well.survival ,19).This review summarizes published results on the expression of CaBPs in mammalian retinas. One of the main purposes of this summary was to see whether CaBPs can serve as type-specific markers of retinal neurons for experimentation. In certain cases, CaBPs are expressed consistently by members of a single (or a couple of) cell type, such as in the case of REC for type 2 mouse BCs and human DB2 cells. However, this appears to be a rare case and the rest of the CaBPs included in this study, particularly the ones in focus of this review , are expressed by heterogenous neuron populations in both the mouse and the human retinas . In these latter cases, the CaBP-expressing outer retinal neurons  have been identified morphologically as well (see the above descriptions). However, in the case of the mouse and human inner retinal neurons, the picture seems to be more complex. While certain cell types have been identified, including the CaB/CaR-expressing mouse starburst cells and the CaR/CaB/PV-expressing AII cells in the human retina, the identity of many other CaBP+ is unknown in both species. It is obvious that further experimentation is needed to fill the remaining gaps.At the same time, the identities of PV+ mouse ACs and CaR-, CaB- and PV-expressing human GCs are poorly studied. Once a comprehensive characterization scheme becomes available for both species, CaBPs may prove to be suitable as type-specific markers for inner retinal neurons as well. However, the heterogeneity of CaBP-labeled populations necessitates the application of a second (or third) neurochemical marker and/or the study of certain morphological characteristics as well to sort the labeled populations into groups of distinct types and possibly subtypes. There are two additional caveats concerning the issue of CaBPs as specific neuronal markers in the retina, with the first one being the reliability of the applied markers and by extension, the recorded signal. As we outlined above, circadian rhythmicity, for example, can alter PV expression in the rat retina  and thus2+ binding by PV and CaB/CaR may modify the response kinetics by altering the duration of different phases of the evoked intracellular Ca2+ increments. In this scenario, the presence of intracellular PV should buffer later phases (> 100 ms) of Ca2+ waves, thereby making neuronal responses more transient. On the other hand, CaB and CaR (being relative fast Ca2+ buffers) chelate intracellular Ca2+ from the beginning of light-evoked Ca2+ waves, meaning that the transient response component would be truncated as well, which would eventually lead to more sustained responses in turn. CaB has also been shown to interfere with the Ca2+-dependent inactivation of Ca2+ currents as well as further slowing down the response decay, thus prolonging light-evoked responses. Whether this functional aspect is relevant for retinal neurons or not has not been explored in depth. In certain cases, such as PV+ mouse alpha GCs and human parasol cells, the hypothesis might seem relevant as both GC populations tend to respond with transient spiking to light flashes. Moreover, PV seems to be a more specific marker for inner retinal neurons, where AC and GC light responses tend to be more transient than the neurons of the outer retina. HCs, for example typically provide sustained responses and they rarely express PV in mammals. On the other hand, CaB is typically expressed by many outer retinal cells, such as PRs, HCs and BCs , that very often maintain sustained light responses. Sustained neurons exist among inner retinal ACs and GCs as well, and so do CaB- and/or CaR-expressing cells. Therefore, this functional expression of CaBPs that determines neuronal response kinetics might as well be a line of research worth pursuing in the mammalian retina.The second aim of this review was to see whether the expression of CaBPs determines the excitability and/or activity patterns of retinal neurons. These physiological parameters have often been measured by examining their light stimulus-evoked membrane potential changes and action potentials possibly generated upon this activation. As discussed above, the different kinetics of CaThe third and last aim of this review was to compare CaBP expression in the mouse and human retinas to determine the applicability of collected mouse data for future studies on the human retina. In this regard, one can conclude that there are major differences between the two species , Table 1


Very little is known about where our pet rabbits come from: Who the breeders are, how good/or bad the conditions are that breeding rabbits are kept in, or whether breeders are being monitored by local authorities. This study aimed to bring to light information on breeding rabbits and breeders in the UK. Several methods of data collection were used combining data from online sales adverts, with a breeder survey and a council freedom of information request. From 3446 online rabbit sale adverts we found 94.5% of adverts were from England and only 1% of breeders were licenced. Out of 33 breeders surveyed, 51.5% provided smaller housing than recommended and housed most rabbits singly, against recommendations, and males were most likely to be housed singly, in too small conditions. However, most provided toys and a diet compliant with recommended guidelines. The most commonly sold/bred rabbits were breeds with flat-faces, which can cause significant health and well-being problems. A freedom of information request sent to 10% of UK councils revealed inconsistency in licensing conditions and confusion about eligibility. Without appropriate guidelines for housing and husbandry and regulation, rabbits within the pet rabbit breeding industry are at risk of compromised welfare. Conditions of pet rabbit breeding colonies and breeder practices are undocumented and very little is known about the pet rabbit sales market. Here, multiple methods were employed to investigate this sector of the UK pet industry. A freedom of information request sent to 10% of councils revealed confusion and inconsistency in licensing conditions. Data from 1-month of online sale adverts (3446) identified 646 self-declared breeders, of which 1.08% were licensed. Further, despite veterinary advice to vaccinate rabbits from five weeks, only 16.7% rabbits were vaccinated and 9.2% of adult rabbits were neutered. Thirty-three breeders completed a questionnaire of which 51.5% provided smaller housing than recommended, the majority housed rabbits singly and bucks were identified as most at risk of compromised welfare. However, most breeders provided enrichment and gave a diet compliant with recommended guidelines. Mini-lops and Netherland dwarfs were the most commonly sold breeds, both of which are brachycephalic, which can compromise their health and wellbeing. From sales data extrapolation, we estimate that 254,804 rabbits are purposefully bred for the UK online pet sales market each year. This data is the first of its kind and highlights welfare concerns within the pet rabbit breeding sector, which is unregulated and difficult to access. Rabbits are the third most popular pet in the UK, with an estimated 1 to 1.5 million population . Howeverhttps://thebritishrabbitcouncil.org/) provides housing advice, but this does not include detailed guidance on meeting the welfare needs of a rabbit breeding colony. Therefore, breeders must refer to guidelines for pet rabbits given by the Welsh and Scottish governments [No specific guidelines exist for the housing and husbandry of rabbits bred for the UK pet trade. The British Rabbit Council  A suitable environment to live in; (2) a suitable diet; (3) to be able to express normal behaviour; (4) to be housed with, or apart, from other animals; (5) to be protected from pain, suffering, injury and disease . HoweverAmongst pet rabbits, targeted surveys have revealed numerous welfare issues associated with pet rabbit housing and husbandry. According to the 2018 PDSA  Animal Wellbeing (PAW) report, which surveyed over 400 rabbit owners, most pet rabbits were purchased from pet shops or garden centres 36%), 17% were acquired from rescue/re-homing centres, and 15% of rabbits were from a friend, neighbour or family member 6%, 17% w,13,14,15Two recent papers have demonstrated the utility of collecting data from online pet sale adverts for shedding light on pet relinquishments ,17, yet To identify the most common breeds, location of breeders, vaccination/neuter status at point of sale and self-declared licence information from data gathered from online sale adverts.To establish the housing and management conditions of pet rabbit breeding colonies via online questionnaire.To establish the consistency of licensing and welfare assessment of UK pet rabbit breeding colonies by local authorities.The aim of this study is to fill an important gap in knowledge about the conditions of rabbits in the pet breeding trade and identify any specific welfare concerns. The overall aim of the study will be met by the following objectives:As so little is known about the housing conditions of breeding rabbits, results will be compared to sources of legislation and guidelines for laboratory, farmed and pet rabbits. This study will provide important preliminary data on an opaque sector within the pet industry, the results of which would be relevant specifically to pet rabbit welfare in the UK, but will also highlight issues of relevance in any country where rabbits are kept as pets. Such work could lead towards further research and the formation of guidelines for the housing and care of pet rabbit breeding colonies, which could have a significant impact on rabbit welfare.This research has been approved by the University of Winchester and the University of Nottingham ethics boards. The rabbit breeder questionnaire was anonymous and positive consent to participate was gained from all questionnaire respondents.A random sample of 40 councils (10%) in England, Scotland and Wales was selected from a total of all 407 councils via a random number generator from a list on the Local Government Information Unit . The geoA second FOI request was sent to the same 10% of councils on 25th May 2018 to gather the same data as above for the year of 2017 and to include the number of renewal applications received for rabbit breeders in 2016 and 2017, again councils were re-contacted two weeks after the cut-off date of 20th June 2018.www.pets4homes.co.uk) in a 1-month period (26th June 2018–26th July 2018) were collected and analysed. Pets4Homes was chosen to collect data from as it is the largest pet only classified advert website in mainland UK . Data collected for each advert included: Location (town and county), breed, seller username, advertiser type (private seller or breeder/frequent advertiser), age of rabbits for sale, whether the advertiser deemed them ready for sale, whether the breeder was council licensed, and microchip, neutering and vaccination status. Data was collected via web-scraping using the rvest package [http://www.rstudio.com/) and stored in an Excel spreadsheet for analysis.To estimate how many rabbit breeders are currently active within the UK and gather details on the rabbits being sold all adverts posted on the Pets4Homes website . Only one pet sale website was used to avoid sending multiple emails to the same breeder. In order for the questionnaire to reach the targeted responders, those contacted from the pet sale website were those specifically listed as a ‘breeder’.To widen the reach of the questionnaire, the RWAF was contacted to ask if they held a list of rabbit breeders, however they did not. The British Rabbit Council (BRC) was contacted on 24 June 2017 to ask if they possessed any guidelines on housing for breeding colonies of rabbits sold to the pet trade. They responded that they held no such information. The questionnaire was also sent via email to 12 individual breeders on the BRC directory whose email addresses were listed and to breeders whose contact details were found on the Pets4Homes website  received and approved a new pet shop licence application from a rabbit breeder and received one renewal application, and Amber Valley Borough Council received a renewal application which was visited by an inspector; 36 of these 40 councils reported no licensed rabbit breeders in 2017. The online sales data collected in 2018 from 26th June to 17th July revealed seven breeders who said they were licensed, one each at two of the councils surveyed by the FOI request: Amber Valley Borough Council and East Hampshire District Council; and one each at five additional councils: Manchester City Council, North Lanarkshire Council, Northumberland County Council, South Lakeland District Council and Wakefield Metropolitan District Council.In response to the open question in the 2017 FOI request ‘Could you please detail your procedure for approving pet shop licenses for rabbit breeders that sell for the pet trade?’, one council stated that “sales made to the trade and not direct to the public do not require a pet shop licence”, another said that a licence was needed if selling animals from a commercial premises or via the internet, and a third council said that all pet shops selling rabbits needed to keep a register of where rabbits were purchased i.e., suppliers and where they were sold. With regards to licensing procedure, six (15%) used veterinary input to inform decisions on licence approval, 10 (25%) inspect the premises that the licence has been applied for, six (15%) used guidance of legislation to inform licensing, 13 (32.5%) stated they did not have a specific procedure and six (15%) considered the question not applicable .In the four weeks from 26th June 2018 to 17th July 2018, 3446 unique adverts on Pets4Homes were identified for rabbits for sale. Amongst these, were 646 self-declared breeders posting 1910 adverts, of which only seven breeders, just 1.08%, were council licensed. A total of 173 breeders were within the council areas where the FOI requests were sent see Table A1The most common breeds advertised were Mini-lop (39.3%), mixed breed (14.5%), Netherland Dwarf (13.8%) and Lionhead (9.7%)  for a group of three laboratory rabbits over 10 weeks of age weighing 2.5–3 kg [Singly housed rabbits had the lowest median housing size, with bucks having the smallest median housing size overall . All gro2.5–3 kg .2 in farmed rabbits [2), 17 breeders did not meet these for some or all of their rabbits. Four breeders did not indicate housing height for all rabbits, and 21 breeders (63.6%) provided housing height less than the 75 cm recommended by the Scottish Government for pet rabbits [Adult bucks and does have a recommended floor area of 7500 cm rabbits , 10 breebreeders 3.6% prov rabbits ,9. All hBucks were housed singly by breeders more often than any other type of rabbit (76.7%), and group housing was rare with only three breeders housing bucks in pairs and three housing bucks in groups, two of which were in a bachelor colony.For does without kits, 49.5% housed them singly, 24.2% housed some singly and in pairs, 9.1% housed them in pairs, 9.1% housed them in groups and 6.1% did not specify how does were housed. For does with kits, 33% (11) housed does with kits in groups with 67% housing them singly. Breeding does had a mean breeding lifetime of 24.4 months  with a mean starting age of 7.8 months  and an aAll but one breeder handled their kits with 85% (28) starting handling between birth and leaving the nest. Out of the breeders who handled their kits, all except one handled kits at least daily (91%). Of all breeders, one took into consideration whether the doe was happy with handling before handling the kits.Breeders sold kits directly to the general public (75.8%), pet shops (18%), as show animals (3%) and to other breeders (15%). Three breeders (9%) indicated that they sold kits online. Two breeders did not specify who they sold their kits to (6.1%) and three (9.1%) did not specify the age at which they were sold. The average minimum age when kits were sold was 8.5 weeks  with two breeders selling their kits before the recommended minimum threshold of eight weeks .All breeders indicated that they gave access to fresh grass for all their rabbits, however when asked to detail their feeding regime, only four breeders included fresh grass. In compliance with the RWAF guidelines ; approp2).Rabbits were allowed access to runs by 31/33 breeders (93.9%). One breeder specified that rabbits had access to runs several times daily but did not give run measurements. Two breeders specified “free roam” or “large runs” but did not give specific measurements. Access to runs was variable dependent upon doe status (litter or no litter), age and sex but daily access to runs was the most common . Only a With the exception of one, all breeders provided all rabbits with environmental enrichment (97%). Enrichment varied, with balls being the most common enrichment provided by 58% of breeders. Chew toys were the next most common enrichment given by 36.4% of breeders, followed by cardboards tubes (30%) and boxes and hay racks (24.2%). Only twelve breeders (36.4%) provided hiding places in the form of boxes or tunnels. Foraging behaviour was encouraged by 45.5% of breeders by providing treat filled boxes/balls or hay filled logs or tubes.The aim of this study was to fill in a gap in knowledge about the conditions of rabbits in the pet breeding trade and the demographics of the UK rabbit sales market. The following objectives were fulfilled in order to meet this aim: An online questionnaire investigating the housing and husbandry of UK breeding rabbits was distributed via social media; results from 33 breeders were compared to existing guidelines/legislation for laboratory, farm and pet rabbits. The current status of licensing of rabbit breeders in 10.3% of UK  councils was established. One months’ worth of data from online advertisements was collected from the Pets4Homes website detailing the rabbits for sale and the status of these rabbits including; age, breed, location, council license status, neuter and vaccination status and seller type. This information can be used to estimate the newly bred rabbit population in the UK, the most common breed types, age at which kits are sold and location of breeders with relation to the council areas sent an FOI request. Information such as this is important to collect as the status of rabbits being bred for sale is an unknown area and this data provides the only known overview of pet rabbit breeding. It also provides a comparison to the council data for how many breeders are currently licensed compared to how many self-declared breeders were actively selling rabbit kits.A recent paper from Neville et al.  identifiTo our knowledge, there were no published accounts identifying the most common rabbit breeds in the UK prior to this work. This may be because it has been deemed information that is not important, but with the rise of welfare problems due to breeding e.g., brachycephalism (shortened skulls) in dogs , cats, a2, minimum height 75 cm) [The Scottish and Welsh guidelines on rabbit welfare from 2018 and 2009, respectively, provide guidance consistent with each other and the RWAF on suitable rabbit housing  ,7. Such In our breeder questionnaire the majority of breeding rabbits (49.5% to 76.7% depending on rabbit type) were housed singly, with just 10 breeders housing rabbits in pairs. Singly housed adult rabbits had the lowest average floor area, with bucks having the smallest overall housing. In many cases single bucks’ housing was smaller than single does’ and six breeders (18%) who housed their rabbits singly had floor areas below the minimum recommended for laboratory rabbits. With regards to height requirements for housing 3% of breeders housing was below laboratory requirements and 61% was below pet requirements; this limits behavioural expression, which can result in abnormal and/or stereotypic behaviour . Singly In addition to being housed in the smallest areas, bucks were housed singly by breeders more often than any other type of rabbit (76.7%). There is no ecological reason to justify housing bucks in smaller areas or singly more than does, yet our finding supports that of the 2018 PDSA report, which found that owners of male rabbits are significantly more likely to house them alone (67%) than owners of female rabbits (35%) [Although some breeders may purchase specified breeding rabbit housing, it is likely that many breeders will purchase commercially available pet rabbit housing. The ability to purchase hutches and runs that do not meet minimum welfare standards needs to be addressed. Minimum requirements for optimum welfare, such as those stipulated by the Welsh and Scottish governments should be implemented across the UK and accommodation that does not meet these requirements should not, in our opinion, be sold. Further, there is a need to develop guidance specifically for rabbits kept for breeding, which is currently overlooked by all guidance excepting that given for rabbits used for scientific and meat purposes.Dental disease is the most common problem described by vets in UK domestic rabbits, which includes breeding rabbits. Rabbits’ teeth grow continually, thus the correct diet high in fibre from hay or grass needs to be fed in order to wear the teeth down adequately and prevent dental disease. Abnormal wear of the teeth leads to malocclusion, which if unnoticed can result in serious health and welfare issues, including death . Here, rAlmost all breeders surveyed here provided enrichment for their animals, and the majority of enrichment was food orientated to promote foraging behaviour indicating that breeders have some knowledge of their animals’ welfare. However, ecological relevance has been shown to be of great importance in selecting enrichment material for captive animals . Most brIn order for authorities to be able to enforce the animal welfare laws outlined in the Animal Welfare Act (2006)  they mushttps://rabbitwelfare.co.uk/campaigns/capone-campaign/) where the charity have committed resources to monitor online rabbit sales and report irresponsible unlicensed sellers directly to councils and the RSPCA in order to make authorities aware of the problems caused by unlicensed pet sales [The random sample of councils contacted here received one license application for rabbit breeders in 2016  and one license application in 2017 that was inspected (East Hampshire District). A total of seven self-declared breeders advertising on Pets4Homes in June-July of 2018 reported to hold a council license, of which one was in Amber Valley District Council and one East Hampshire District suggesting that these councils renewed the licenses in 2018 also. Considering the 2018 estimate of seven breeders being licensed in the UK would mean that only 1.08% of self-declared breeders selling directly to the public were actually licensed to do so. These results suggest that the majority of the current population of more than one million UK pet rabbits must be bred by unlicensed, and therefore unregulated, breeders. This is a problem, as it means that there are no animal welfare safeguards in place for 98.92% of the UK rabbit breeding population (such as site inspections and maintained records), and no way to know where the animals are or to make irresponsible breeders accountable. For this reason the RWAF have launched the Capone Campaign  (England) Regulations 2018 , which gOur results suggest that there are prevalent welfare issues within the UK pet rabbit breeding sector. Although only a small percentage of rabbit breeders took part in this study, significant diversity was seen in housing and husbandry conditions available to breeding rabbits. Housing and run size was variable, with 18.2% of bucks and 6.1% of does housed in restricted conditions smaller than the minimum recommended cage size for laboratory rabbits, and 33% of breeders housed rabbits in hutches smaller than the minimum recommended housing size for pet rabbits. Furthermore, social requirements of breeding rabbits were seldom met by UK rabbit breeders with the majority of bucks and does being housed singly. Diet and enrichment were two areas that presented the least welfare concerns from rabbit breeders; the majority of breeders showed knowledge of the need for hay in rabbits’ diet, however just over half of breeders failed to increase nutritional provisions to meet the needs of pregnant and lactating does, meaning there is room for improvement. Enrichment was provided by all but one breeder; however it is unclear whether breeders provided the right types of enrichment to fulfil their rabbits normal and natural behaviour needs with the most common enrichment being a non-ecologically relevant ball and 63.6% of breeders failing to provide ecologically relevant enrichment in the form of hiding places.Online sales data implies that most rabbit kits are sold un-vaccinated, despite veterinary advice to vaccinate from five weeks of age. Further, we have been able to extrapolate the first estimate of the rabbit size of the rabbit breeding market, with a possible 254,804 rabbits purposefully bred for the UK online pet sales market each year; the majority which appear to be bred in England. The most commonly sold rabbit breeds were identified as being Mini-lops and Netherland dwarfs; both breeds with brachycephalic conformations, which can have devastating health consequences.The data collected here could be of value for organisations to implement interventions to safeguard the welfare of breeding rabbits. Council licensing could be used as a safeguard, through which the Animal Welfare Act (2006)  could beThe variability in practices by breeders may be due in part to the distinct lack of guidance available to clearly detail standards for providing optimum welfare amongst rabbit breeding colonies. Therefore, if the housing and husbandry conditions, and thus welfare of breeding rabbits in the UK is going to improve it is paramount that consistent, standardised guidance is provided to rabbit breeders and that appropriate, clear legislation is created regarding eligibility for licensing.


We report Polarization-resolved Second Harmonic Generation (P-SHG) and directional SHG  measurements of equine foetal and adult collagen in meniscus, over large field-of-views using sample-scanning. Large differences of collagen structure and fibril orientation with maturation are revealed, validating the potential for this novel methodology to track such changes in meniscal structure. The foetal menisci had a non-organized and more random collagen fibrillar structure when compared with adult using P-SHG. For the latter, clusters of homogeneous fibril orientation (inter-fibrillar areas) were revealed, separated by thick fibers. F/B SHG showed numerous different features in adults notably, in thick fibers compared to interfibrillar areas, unlike foetal menisci that showed similar patterns for both directions. This work confirms previous studies and improves the understanding of meniscal collagen structure and its maturation, and makes F/B and P-SHG good candidates for future studies aiming at revealing structural modifications to meniscus due to pathologies. Degradation of the meniscal tissue can increase articular cartilage strain2, and may lead to cartilage degeneration and osteoarthritis3. Knowledge of the complex structure of the meniscal extracellular matrix (ECM) has increased thanks to emerging technologies for in situ imaging of intact specimens, such as Optical Projection Tomography (OPT)4. In particular the arrangement of meniscal fascicles4, its tie-fiber organization5, and the menisco-tibial ligament insertion transition have all recently been revealed by investigation of bovine samples6.The meniscus is a semilunar fibrocartilaginous structure interposed between the femoral condyle and the tibial plateau in the knee joint. The meniscus is essential for load transmission across the articular surfaces, for femorotibial joint stability and for long-term joint health7. However, important differences exist: it is a coherent process sensitive to the phase-matching conditions where the measured signal arises from constructive/destructive interferences, it is also instantaneous and free from photobleaching as the signal conversion is due to a structural arrangement and does not involve electronic transition8. SHG microscopy has been used to image fibrillar collagen in specimens including type II collagen in articular cartilage16. Furthermore, because of its coherent nature, the detection of the signal in the direction of propagation (forward - F) provides different imaging features compared to the backward (B) direction17. The F/B ratio increases with the level of homogeneity of noncentrosymmetric structures within the focal volume and has been related to the size of the collagen fibrils for collagen rich tissues19.SHG microscopy is a recent and powerful technique to image the structure of biological specimens as it provides submicron spatial resolution, has low phototoxicity and a high depth selectivity and penetration. In this respect, SHG imaging is similar to multiphoton-excited fluorescence microscopy23. SHG microscopy has revealed a variety of meniscal structural features including: the fiber bundle organization24, the arborization of the tie-fibers5 and the interaction of primary cilia with the ECM25 in healthy menisci. The effect of degradation on ECM density and cell accumulation26, of cyclic loading27 and the meniscal integrity following repair20 have also been imaged by SHG microscopy. Furthermore large images of the whole joint (cartilage and meniscus) were imaged by multiphoton fluorescence and SHG28, and even in-vivo and minimally processed joints using SHG microendoscopy29. SHG and Coherent Anti-Stokes Raman Scattering (CARS) microscopy have also been employed to characterize artificial meniscus-like implants30. However, up to now, the local orientation of the meniscal collagen structures has been mainly characterized by OPT, only to a resolution of ~15 µm permitting identification of the collagen fascicles but not of the individual fibers4. These fibers were later imaged by SHG microscopy in some areas from bovine samples5, but their orientation was discussed only in terms of the SHG intensity. The latter is subject to interference due to the coherent nature of the process, that can result in masking the real underlying structure31 therefore preventing imaging of the fibril orientation and nonlinear tensor properties on its own32. Polarization-resolved SHG (P-SHG) was developed to override this limitation, and has been shown to accurately reveal the alignment distribution of collagen fibrils in various tissues such as tail tendon or skin34, but has never been reported in meniscus.The complex noncentrosymmetric structural organization of meniscal collagen has been previously imaged using Second Harmonic Generation (SHG) microscopy35. The propagation of collagen II deposition has been investigated in the mouse (using an anti-collagen II bioclone) from the inner to outer border, during meniscal growth from foetus to adult36. Furthermore the relative area of meniscus versus articular cartilage has also been characterized during human foetal gestation37. In addition, the meniscus has been shown to increasingly develop cartilaginous properties during maturation38. A recent study also compared vertical meniscal sections in bovine joints from foetuses and adults39, and reported that the spacing between circumferential bundles of fibrils is much higher in foetuses than in adults.The influence of age on the mechanical response of meniscal connective tissue has been studied at the nanoscale in human menisci using atomic force microscopyHere, we demonstrate the unique potential of SHG and P-SHG to measure the collagen structure in the meniscus, and we use these two techniques together with histology to characterize the structural differences between equine foetal and adult meniscal ECM. Understanding and imaging the meniscal ECM could provide new knowledge to support studies on meniscal degradation events in joint degeneration, and potentially new insights towards meniscal regeneration in the future.5. Additional images of all foetal and adult menisci can be found in the Supplementary information  are presented in Figs. The SHG images of Fig. et al.40 was not here necessary to obtain a proper backward image.In the case of mature specimens  was averaged first over the whole image in Fig. Moreover, the differences between the fibril orientation distributions observed in Figs. 41, is the principal component of the adult meniscal ECM (70%), of which 60% is type II and 40% type I42. The ordering of the collagen fibrils within the focal volume of excitation  is usually probed by P-SHG using the anisotropy parameter ρ43 (see Methods section for definition), but this method has been mainly applied to tissues composed of only one type of collagen. Indeed, despite having similar sequences of amino-acids44, collagen I and II have different properties: they are biochemically different, and their fibrils have different sizes12. It is thus a challenge to measure structural parameters such as ρ in such tissues. In previous studies of cartilage tissues, Romijn et al.44 have differentiated collagen I and II but only at some specific pixels, resulting in a ρ mapping with a relatively high level of uncertainty44. This discrimination is also challenging because the average ρ in collagen I and II was found to be similar44.Collagen, which plays a central role in the architecture of the tissue44. As a result, the ρ values averaged over the whole images were undistinguishable between our samples of foetal and adult menisci (not shown here), despite clear different features observable in the images. Overall, the shape and contrast of the polarimetric diagrams extracted at each pixel cannot be exploited due to these complex effects. This comes at a high cost since this is usually the main feature exploited in P-SHG. For instance, this precludes the classification of these tissues by the method previously described by Rouède et al.41, that mainly relies on ρ.Moreover, the semilunar and concave form of the meniscus , which shows that these maps are not sensitive to the precise value of the susceptibility tensor components, and that P-SHG’s ϕ provides a robust way to measure these orientation maps. The ϕ-map in P-SHG Figs.  and 4 prSHG. The backward SHG reveals smaller or more random structures in a complementary way17. The F/B ratio is then smaller for the more disorganized structures - or for structures organized on a size smaller than λSHG17 - and increases with the fibril diameter, or with the diameter of bundle of fibrils of same polarity45. This ratio is averaged over the whole field-of-view and thus provides global information on the collagen bundles in terms of size and arangment. Specifically, it is expected to be smaller for type II collagen, which is known to form fibrils with smaller diameters (few tens of nm46) than type I collagen (few hundreds of nm)48. Collagen II is also present in low amounts in foetuses, but increases with age38. In addition, tie-fibers in mature samples have higher quantities of it compared to the other regions like fascicles49. We therefore expect that the tie-fibers exhibit a lower F/B ratio than the rest due to the type II collagen fibrils present in this structure: accordingly, we have observed that the tie-fibers were the main visible patterns in the backward direction. However, the adults that contains more tie-fibers than foetuses would have a smaller global F/B ratio. This confirms that F/B SHG cannot unambiguously discriminate between type I and type II collagen or measure their respective quantities. Nevertheless, it demonstrates that it is a useful structural probe – complementary to histological assessment – sensitive to the different spatial organizations of the collagen (type II or mix type I/II) that varies with age.The study of the forward and backward SHG (F/B) provides an analysis on the directionality of the SHG signal. The forward SHG image shows at each pixel the degree of phase-matching, with a selectivity on the ordered structures , at the size of the SHG wavelength λet al.39 with multimodal multiphoton microscopy (SHG and two-photon fluorescence), using a local thickness measurement: the inter-fibrillar area reduces with maturity in meniscus. However, it is more precise to use P-SHG to circumvent interference pattern artifacts, as mentioned in the introduction. It is also noteworthy that the change from a random collagen meshwork to a more organized arrangement with maturation - that we have observed here in meniscus - has also been demonstrated in developing cartilage50. Additionally, cartilage maturation has been shown to be coupled with the apparition of different zones51, which is consistent with the formation of clusters of fibrils in meniscus that we have observed in this study.Interestingly, the result of Fig. 5: relatively thick tie-fibers oriented radially, interlacing some fascicles oriented circumferentially. Moreover, this organized structure is not identified in the foetal meniscus: the latter is composed of either a random fibril organization, or no network at all with only parallel fibrils. This study confirms the importance of SHG microscopy, especially the F/B as a structural probe for the study of the meniscal – and potentially the cartilage’s – ECM, as well as the capacity of P-SHG to map the fibril orientation even with a mix of different collagen types. Both techniques are shown to be automated and applicable within a reasonable acquisition time, enabling large scale studies in the future. They also provide quantitative results which can be significant for further studies of the structure of meniscus at the micron-scale. We anticipate their future use for characterization of degeneration in the meniscus or cartilage, and overall furthering the understanding of such tissues.To conclude, we report, for the first time to our knowledge, the mapping of the in-plane orientation of the collagen fibrils within the meniscal tissues over large ROIs. The observed structure of the collagen network in adult samples is in agreement with what has been previously reported (with other techniques) in bovine menisci53. Adult samples (n = 6) were also studied, and had been banked from a previous study. The femorotibial joints were freed of soft tissue, inspected and the menisci harvested. Menisci were included in the study if the macroscopic appearance of all surfaces was normal (no fibrillations or tears) and if the joint of origin was also macroscopically intact (no cartilage erosions or evident pathology). Sample listing can be found on Table 3  20% for 2 weeks prior to paraffin embedding and subsequent sectioning. Five micron sections were cut  of the Faculté de médecine vétérinaire, Université de Montréal. Tissues were obtained from a local slaughterhouse and from horses donated by their owners for research. The age of equine foetuses (n = 5) was estimated using crown-rump measurements as previously describedsee Fig. . A slicecut Fig.  and thencut Fig.  was imagcut Fig. . This seFive micron sections were also cut and stained with haematoxylin, eosin, phloxine and saffron (HEPS) as well as Picro-sirius red to illustrate the collagen network architecture under polarized light microscopy  software and a 6110 multichannel I/O acquisition card . Images were recorded using 50 µs pixel dwell-time. The sample-scanning allowed to do rather large scans  in just one acquisition (no mosaic reconstruction) and in a reasonable acquisition time (55 sec), providing that the scan parameters are optimized and the synchronization between the motor and the acquisition DAQ card is controlled. Each image was acquired 3 times to perform an average measurement. These acquisition parameters were the same for the two techniques F/B SHG and P-SHG. The average power on the sample was adjusted to 20 mW, corresponding to roughly 0.3 nJ/pulse. Raw data visualization was performed with FIJI-ImageJ (NIH55) and image processing was performed with MatLab (The MathWorks). All the images are presented with the full imaged areas, and oriented with the femoral surface on top and the outer surface on the left  delivering ~150 fs pulses at 810 nm and 80 MHz rate was used as the laser source. The microscope is a modified commercial laser-scanning setup , where a plane mirror was inserted before the objective to by-pass the laser-scanning, and send the beam directly to the objective to perform sample-scanning with a translation stage . An achromatic telescope was used to re-size the beam to overfill the back aperture of the objective . The polarization was controlled by a multi-wavelength half-wave plate , and by an achromatic quarter-wave plate  placed before the microscope see Fig. . A mecha17 by taking the median of the pixel-wise F/B signal in the whole field-of-view, the forward and backward images being the average of the 3 acquired frames.A circular polarization of excitation was used to image all structures independently of their orientation in the image plane. A long pass dichroic mirror  placed at the back-focal plane of the objective was used to reflect the SHG generated in the backward direction (epi). This dichroic mirror is translatable, and was inserted only when backward imaging was performed (using circular polarization), not for linearly polarized excitation. To calibrate the F/B measurements i.e. to take into account that the detection efficiency is different in forward and backward directions, we used the isotropic emission of the two-photon excited fluorescence signal from Coumarin 440 (diluted in ethanol), because its fluorescence (400–460 nm) lies partly in the range of the SHG filters (405 ± 5 nm). The obtained ratio between forward and backward directions was 0.4. The F/B ratio was calculated by an established methodet al.56, using a modified version of their MatLab code57. This enables to use a varying linear polarization (from 0 to 180°) that stays linear even if the waveplates are placed before the input of the commercial microscope. It is worth noting that the dichroic used in F/B SHG measurements was removed for P-SHG, and that sample-scanning avoids any other source of polarization distortion compared to the standard laser-scanning. As we performed the SHG measurements, images were acquired for 18 polarization states, spaced by steps of 10° so that the full range of  was covered (the rest of the polarizations being redundant).A perfect linear polarization is needed for P-SHG, so the polarization was calibrated with the routine developed in Romijn et al.58. The collagen fibrils are here assumed to have cylindrical symmetry (C∞ group), and we neglect the chiral components of their nonlinear susceptibility tensor χ(2). Furthermore, in the case where the fibrils are distributed around the imaging plane (which is reasonable for cartilage-like tissue), the out-of-plane orientation δ should be considered (δ = 0 meaning in the plane)44. Even if the Kleinman symmetry should here apply as NIR wavelengths are used to drive the SHG process, thus having energies far from any transition44, this condition is not assumed a priori to be able to verify it in data treatment. The independent non-zero components of the χ(2) are then χ33, χ31 and χ15. The X axis in the XYZ frame corresponds to the same direction as the x axis in the xyz frame. The simplified expression for the two components of the SHG intensity (X being the direction of propagation) can then be written as44:A = χ31/χ15 and B = χ33/χ15cos2δ + (2 + χ31/χ15)sin2δ44, so that ρ0, i.e. that the Kleinman condition applies and that the collagen lies in the imaging plane. Here, we process the P-SHG data with or without a filter that imposes abs(A) < 1.1, as in Romijn et al.44 . We still expect to be close to the Kleinman symmetry (i.e. A ≈ 1) as already mentioned. ρ is in this case the anisotropy parameter in the frame of the tilted fibril, which can be written as ρ = ρ0cos2δ + 3sin2δ where ρ0 is the anisotropy parameter for no tilt58. Summing the above intensities and developing the sine and cosine gives58:0 to ρ43. A custom MatLab routine was then used to extract the relevant information from the P-SHG images. Briefly, a spatial FFT algorithm with respect to the angle θ is used to compute the Fourier transform (variable Ω) of the measured intensity59:For a complete description of P-SHG, see Teulon 2 (resp. ϕ4) of the relative orientation ϕ could then be extracted from the first exponential: 4, a simple arctangent must be used to avoid a wrong re-casting59, ϕ4 which is then in . Putting 2 + (1 − β)(ϕ4+mπ/4) gives the most accurate result59, with m in {−2, −1, 0, 1, 2} calculated to minimize the quantity 59.The estimation ϕ60.The values of orientation ϕ obtained in P-SHG are circular, so their histograms are better represented on polar plots. The orientation is measured, but the polarity remains unknown so the values of ϕ are obtained in . To plot the histogram in a full circle, the values of ϕ were thus duplicated by a central symmetry (which makes the polar histograms inherently centrosymmetric). The orientations are color-coded, as indicated on the histograms . The standard deviation (std) for the values of ϕ must also use a circular calculation, otherwise the boundaries of the distribution are not considered equal and continuous, as an angle should beThe Shapiro-Wilk’s test was first used to reject the normality of the distributions, and the difference was then tested using the Wilcoxon-Mann-Whitney test. The significance threshold was set to 0.05. Meniscus shows regions of low SHG signal meaning discontinuities of the visible collagen structure, attributed to either out-of-plane fibrils or zones with no collagen. The relative surface of these inter-fibrillar areas can be calculated in ImageJ: a threshold (ImageJ “Yen white”) was first applied to the backward SHG images to discriminate the fibrillated areas from the rest (see the masks in Fig. Supplementary information


In particular, JMV5656 increased cytoplasmic Ca2+ levels by a PLC-dependent release of Ca2+ from the endoplasmic reticulum. STIM proteins and Orai Ca2+ channels were activated and played a crucial role. In fact, treatment of the cells with U73122 and thapsigargin modulated the increase of intracellular Ca2+ levels stimulated by JMV5656. Moreover, in RAW264.7 cells intracellular Ca2+ increases did not occur through the binding of JMV5656 to the C3a receptor, since the increase of intracellular Ca2+ levels induced by JMV5656 was not affected by specific siRNA against C3aR. In summary, our study provides new indications for the downstream effects of JMV5656 in macrophages, suggesting that it could activate receptors different from the C3aR.TLQP-21 is a neuropeptide which has been implicated in regulation of nociception and other relevant physiologic functions. Although recent studies identified C3a and gC1q receptors as targets for TLQP-21, its intracellular molecular mechanisms of action remain largely unidentified. Our aim was (i) to explore the intracellular signaling pathway(s) activated by JMV5656, a novel derivative of TLQP-21, in RAW264.7 macrophages, and (ii) to assess linkages of these pathways with its purported receptors. JMV5656 stimulated, in a dose-dependent fashion, a rapid and transient increase in intracellular Ca TLQP-21 (VGF556-576) is one of the best-studied and characterized VGF-derived fragments, and regulates different biological processes, like energy balance, lipolysis, and gastric functions, as well as reproduction and inflammatory pain  levels, and to activate an outward potassium (K+) current in microglial cells  in a phospholipase C (PLC)-dependent way, most likely by binding a receptor different from C3aR.Consequently, the purpose of this study was to investigate intracellular signaling pathways activated by JMV5656 in RAW264.7 cells, and to evaluate whether this peptide could bind to C3aR. The data presented here clearly indicate that in macrophages JMV5656 stimulates intracellular Ca(70-77) was purchased from D.B.A. Italia .TLQP-21 (TLQPPASSRRRHFHHALPPAR) and JMV5656 (RRRHFHHALPPAR) were synthesized using conventional solid phase synthesis and then purified on a C18 reversed phase column. Each peptide was purified to a purity of at least 96% by high-performance liquid chromatography (HPLC). Prior to assay, peptides were first dissolved in ultrapure water, and then diluted in Hank’s Balanced Salt Solution (HBSS) to final working concentrations. Cyclosporine A (CsA), thapsigargin (TG), U73122, 2-aminoethyl diphenylborinate (2-APB), SKF-96365, YM-58483, and EGTA were purchased from Sigma-Aldrich . C3a was purchased from Merck Millipore . C3aL-glutamine  under standard cell culture conditions .RAW264.7 murine macrophage cells were cultured in Dulbecco’s Modified Eagle Medium  supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 IU/ml penicillin, 100 μg/ml streptomycin, and 2 mM 2 as previously described  . Changes in fluorescence corresponded to changes in intracellular calcium levels. TLQP-21, JMV5656, C3a, and C3a(70-77) were diluted in HBSS and injected into the wells by an automated injector system. Where indicated, antagonists and inhibitors were added at different times before the end of the incubation with FLUO-4 NW as previously described (F) were normalized against the baseline acquired immediately before stimulation (F0).RAW264.7 cells were plated at 40,000 cells/well into black walled, clear bottom 96-well plates  and cultured 2 days up to 90–100% confluence. Before assay, the medium was removed and cells were incubated with FLUO-4 NW for 40 min at 37°C and 5% COescribed . Fluoresescribed . BrieflyCa3.1 siRNA  (Eurofins Genomics). Transfection was performed in a 12-well or 96-well plate (Euroclone), depending on the type of the experiments, using DharmaFECT 4 Transfection Reagent  according to the manufacturer’s protocol. Subsequent experiments were performed 24 h after transfection.C3aR siRNA duplex  was purchased from Eurofins Genomics . To control for non-specific effects of transfection, the negative control group was transfected with K®ND1000 (Thermo-Scientific). Total RNA was transcribed to cDNA using M-MLV Reverse Transcriptase . cDNA was amplified by PCR using GoTaq®G2 DNA Polymerase  in the presence of the following primers (Sigma-Aldrich): C3aR forward: 5′-CCTTCTCCTTGGCTCACCT-3′; C3aR reverse: 5′-AAATACGGGCACACACATCA-3′; GAPDH forward: 5′-GCCATCAACGACCCCTTCATTG-3′; GAPDH reverse: 5′-TCTGTCATGAGGTTGGCTTTCAG-3′. The amplified samples were loaded in equal volumes onto 1% agarose gel in Tris-Acetate-EDTA (TAE) buffer, and then quantified using a Kodak Digital ScienceTM Image Station 440CF system. C3aR mRNA levels were normalized to GAPDH levels.Total RNA was extracted from RAW264.7 cells using EuroGOLD Trifast reagent (Euroclone) and quantified spectrophotometrically using a Nanodropg for 1 h at 4°C. After 15–20 min of incubation on ice, pellets were resuspended in assay buffer , and quantified by BCA assay (Thermo-Scientific). Equivalent amounts of samples were run on precast 4–12% gradient gels , and transferred to a polyvinylidene difluoride (PVDF) membrane . Non-specific sites were blocked with 5% dried fat-free milk dissolved in PBS supplemented with 0.1% Tween-20 (PBS-T) overnight at 4°C. After washes in PBS-T, membranes were incubated with primary antibodies  for 2 h at room temperature (RT). Membranes were then washed with PBS-T and incubated with a peroxidase-coupled secondary antibody  for 1 h at RT. Signals were developed with the enhanced chemioluminescence (ECL) system  and detected with a Kodak imaging system. Image J software  was used to quantify protein bands. C3aR protein levels were normalized for β-actin levels.RAW264.7 cells were rinsed twice with cold phosphate-buffered saline (PBS) and harvested in cold lysis buffer  containing a protease inhibitor cocktail . Cell lysates were sonicated on ice and centrifuged at 115,000 ×t-test or, when appropriated, by one-way analysis of variance (ANOVA) followed by Tukey’s test. A p-value of less than 0.05 was considered significant.Values are expressed as mean ± standard error of the mean (SEM). Normality of data distribution was assessed by the Jarque-Bera test. Experiments were independently replicated at least three times. The statistical significance of differences between groups was evaluated with Student’s 2+ levels in RAW264.7 macrophages. To this aim, cells were stimulated with varied concentrations of JMV5656, ranging from 1 nM to 10 μM. Our results demonstrate that JMV5656 in the concentration range of 500 nM to 10 μM (EC50: 0.34 μM) caused a significant rise of intracellular Ca2+ levels, reaching a plateau at higher concentrations . The treatment with 2 μM TG did not modify basal levels , since the treatment of RAW264.7 cells with the antagonist U73122 (10 μM) induced a partial, approximately 38%, but significant decrease in JMV5656-induced Ca2+ release from the ER -mediated Ca2+ release from the ER. As shown in Figure 2+ mobilization induced by JMV5656 of about 65% in RAW264.7 cells. Again, neither U73122 nor 2-APB modified basal fluorescence  proteins and Orai proteins  inhibited the rise in intracellular Ca2+ concentration by 71%  peptides in RAW264.7 cells. RT-PCR demonstrated that RAW264.7 cells expressed high levels of C3aR mRNA  (1 μM) induced an increase of intracellular Ca2+ levels  was almost superimposable, we decided to use C3a(70-77) for the next experiments.Recently, C3aR has been identified as the target for TLQP-21 in macrophages and ovary cells . We inveA Figure , and thas Figure . Howevers Figure . Since t(70-77) induced, like JMV5656, a desensitization mechanism of its receptor, RAW264.7 cells were stimulated with repeated administration of C3a(70-77) given at 30 min interval. Intracellular Ca2+ levels rose significantly after the first administration of 1 μM C3a(70-77)   induced a robust increase in cell fluorescence , applied 30 min after JMV5656, induced a lower but significant increase of intracellular Ca2+ levels  bind to two different receptors.In order to investigate whether C3a) Figure . After 3s Figure , suggest) Figure . In facte Figure . Interess Figure  in the c2+ levels mediated by 1 μM C3a of about 38%, while no reduction was observed after 1 μM JMV5656 Ca2+ stimulation  to IP3 leading to a decrease in free cytosolic Ca2+ , while the binding of IP3 to its receptor triggers the release of Ca2+ ions from the ER. Our results demonstrate that the treatment of RAW264.7 cells with 2-APB, an IP3R antagonist, greatly reduced the increase of intracellular Ca2+ levels induced by JMV5656, further confirming that the binding of the peptide with its receptor causes the release of Ca2+ from the ER in a PLC-dependent way.PLCs are known to cleave phosphatidylinositol-4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and IP2+, STIM proteins are activated and translocate to region of the ER in close proximity with the plasma membrane, where they activate Orai Ca2+ channels generating a Ca2+ release-activated Ca2+ (CRAC) current (2+ influx is supposed to be linked to the production of reactive oxygen species (ROS) and to be necessary for the engulfment of apoptotic cells  channels  peptides induced an increase of intracellular Ca2+ levels in these cells. However, unlike JMV5656, repeated administration of C3a(70-77) did not induce a desensitization mechanism, suggesting the existence of two different receptors for these peptides in RAW264.7 cells. Indeed, in our experimental setting, incubation with siRNA against C3aR decreased intracellular Ca2+ mobilization induced by C3a but not by JMV5656, confirming the presence of a receptor different from C3aR, and still unidentified, in RAW264.7 cells for JMV5656. Indeed the RAW264.7 also express significant amounts of mRNA for gC1qR, which has been proposed to mediate the effects of TLQP-21 in macrophages (Recently, C3aR has been indicated as possible receptor for TLQP-21 in rodents . C3aR isrophages . We coulOur research provides new evidence for the downstream effect of the binding of JMV5656 to its receptor in macrophages, and suggests the existence of different receptors for this peptide in rodent cells. Improving knowledge about the intracellular pathway activated by JMV5656 and the identification of the human receptor could help to improve the treatment of several human disorders, including neuropathic pain.in vitro using cell lines only. No experiments were performed on animals or involved human beings. Approval from the local Ethic Committee or other regulatory agencies was not required.The study was made AT, GB, VL, and RO supervised the entire project, designed the research, and wrote the paper. LM, LR, EB, RM, J-AF, and PV conceived and designed the experiments, performed the research, interpreted and analyzed the data. AT, GB, RO, and VL analyzed the data and critically revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.