Neuronal A2A receptor exacerbates synapse loss and memory deficits in APP/PS1 mice.

Early pathological upregulation of adenosine A2A receptors (A2ARs), one of the caffeine targets, by neurons is thought to be involved in the development of synaptic and memory deficits in Alzheimer's disease (AD) but mechanisms remain ill-defined. To tackle this question, we promoted a neuronal upregulation of A2AR in the hippocampus of APP/PS1 mice developing AD-like amyloidogenesis. Our findings revealed that the early upregulation of A2AR in the presence of an ongoing amyloid pathology exacerbates memory impairments of APP/PS1 mice. These behavioural changes were not linked to major change in the development of amyloid pathology but rather associated with increased phosphorylated tau at neuritic plaques. Moreover, proteomic and transcriptomic analyses coupled with quantitative immunofluorescence studies indicated that neuronal upregulation of the receptor promoted both neuronal and non-neuronal autonomous alterations, i.e. enhanced neuroinflammatory response but also loss of excitatory synapses and impaired neuronal mitochondrial function, presumably accounting for the detrimental effect on memory. Overall, our results provide compelling evidence that neuronal A2AR dysfunction, as seen in the brain of patients, contributes to amyloid-related pathogenesis and underscores the potential of A2AR as a relevant therapeutic target for mitigating cognitive impairments in this neurodegenerative disorder.

Exacerbation of C1q dysregulation, synaptic loss and memory deficits in tau pathology linked to neuronal adenosine A2A receptor.

Accumulating data support the role of tau pathology in cognitive decline in ageing and Alzheimer's disease, but underlying mechanisms remain ill-defined. Interestingly, ageing and Alzheimer's disease have been associated with an abnormal upregulation of adenosine A2A receptor (A2AR), a fine tuner of synaptic plasticity. However, the link between A2AR signalling and tau pathology has remained largely unexplored. In the present study, we report for the first time a significant upregulation of A2AR in patients suffering from frontotemporal lobar degeneration with the MAPT P301L mutation. To model these alterations, we induced neuronal A2AR upregulation in a tauopathy mouse model (THY-Tau22) using a new conditional strain allowing forebrain overexpression of the receptor. We found that neuronal A2AR upregulation increases tau hyperphosphorylation, potentiating the onset of tau-induced memory deficits. This detrimental effect was linked to a singular microglial signature as revealed by RNA sequencing analysis. In particular, we found that A2AR overexpression in THY-Tau22 mice led to the hippocampal upregulation of C1q complement protein-also observed in patients with frontotemporal lobar degeneration-and correlated with the loss of glutamatergic synapses, likely underlying the observed memory deficits. These data reveal a key impact of overactive neuronal A2AR in the onset of synaptic loss in tauopathies, paving the way for new therapeutic approaches.

RhoA Inhibitor Treatment At Acute Phase of Spinal Cord Injury May Induce Neurite Outgrowth and Synaptogenesis.

The therapeutic use of RhoA inhibitors (RhoAi) has been experimentally tested in spinal cord injury (SCI). In order to decipher the underlying molecular mechanisms involved in such a process, an in vitro neuroproteomic-systems biology platform was developed in which the pan-proteomic profile of the dorsal root ganglia (DRG) cell line ND7/23 DRG was assessed in a large array of culture conditions using RhoAi and/or conditioned media obtained from SCI ex vivo derived spinal cord slices. A fine mapping of the spatio-temporal molecular events of the RhoAi treatment in SCI was performed. The data obtained allow a better understanding of regeneration/degeneration induced above and below the lesion site. Results notably showed a time-dependent alteration of the transcription factors profile along with the synthesis of growth cone-related factors (receptors, ligands, and signaling pathways) in RhoAi treated DRG cells. Furthermore, we assessed in a rat SCI model the in vivo impact of RhoAi treatment administered in situ via alginate scaffold that was combined with FK506 delivery. The improved recovery of locomotion was detected only at the early postinjury time points, whereas after overall survival a dramatic increase of synaptic contacts on outgrowing neurites in affected segments was observed. We validate these results by in vivo proteomic studies along the spinal cord segments from tissue and secreted media analyses, confirming the increase of the synaptogenesis expression factors under RhoAi treatment. Taken together, we demonstrate that RhoAi treatment seems to be useful to stimulate neurite outgrowth in both in vitro as well in vivo environments. However, for in vivo experiments there is a need for sustained delivery regiment to facilitate axon regeneration and promote synaptic reconnections with appropriate target neurons also at chronic phase, which in turn may lead to higher assumption for functional improvement.

Proteomic Analysis of the Spatio-temporal Based Molecular Kinetics of Acute Spinal Cord Injury Identifies a Time- and Segment-specific Window for Effective Tissue Repair.

Spinal cord injury (SCI) represents a major debilitating health issue with a direct socioeconomic burden on the public and private sectors worldwide. Although several studies have been conducted to identify the molecular progression of injury sequel due from the lesion site, still the exact underlying mechanisms and pathways of injury development have not been fully elucidated. In this work, based on OMICs, 3D matrix-assisted laser desorption ionization (MALDI) imaging, cytokines arrays, confocal imaging we established for the first time that molecular and cellular processes occurring after SCI are altered between the lesion proximity, i.e. rostral and caudal segments nearby the lesion (R1-C1) whereas segments distant from R1-C1, i.e. R2-C2 and R3-C3 levels coexpressed factors implicated in neurogenesis. Delay in T regulators recruitment between R1 and C1 favor discrepancies between the two segments. This is also reinforced by presence of neurites outgrowth inhibitors in C1, absent in R1. Moreover, the presence of immunoglobulins (IgGs) in neurons at the lesion site at 3 days, validated by mass spectrometry, may present additional factor that contributes to limited regeneration. Treatment in vivo with anti-CD20 one hour after SCI did not improve locomotor function and decrease IgG expression. These results open the door of a novel view of the SCI treatment by considering the C1 as the therapeutic target.

Glutamatergic cells in the periaqueductal gray matter mediate sensory inputs after bladder stimulation in freely moving rats.

OBJECTIVES: To determine the phenotype of the ventrolateral part of the periaqueductal gray matter neurons after bladder stimulation. METHODS: In the experimental group, electrical stimulation of the bladder was carried out under freely moving condition by a bipolar stimulation electrode implanted in the bladder wall. Thereafter, the brain sections were processed for immunohistochemical analysis using antibodies against c-Fos (neuronal activation marker) together with one of the following: tyrosine hydroxylase (dopaminergic cell marker), vesicular glutamate transporter (glutamatergic cell marker), serotonin, glutamate decarboxylase (glutamate decarboxylase 67, gamma-aminobutyric acid cell marker) and neuronal nitric oxide synthase. We used design-based confocal stereological analysis to quantify the immunohistochemically stained sections. RESULTS: A significant increase in the number of c-Fos-positive cells in the ventrolateral part of the periaqueductal gray matter after stimulation was found. Furthermore, the ratio of c-Fos cells double labeled with vesicular glutamate transporter was significantly higher in the ventrolateral part of the periaqueductal gray matter region in the stimulated compared with the sham group. Quantitative analysis of the other four cell types did not show any significant difference. CONCLUSION: These findings suggest that glutamatergic neurotransmission in the ventrolateral part of the periaqueductal gray matter is seemingly the main pathway to be activated after receiving sensory signals from the bladder.

Human temporal lobe epilepsy analyses by tissue proteomics.

Although there are many types of epilepsy, temporal lobe epilepsy (TLE) is probably in humans the most common and most often studied. TLE represents 40% of the total epilepsy form of the disease and is difficult to treat. Despite a wealth of descriptive data obtained from the disease history of patients, the EEG recording, imaging techniques, and histological studies, the epileptogenic process remains poorly understood. However, it is unlikely that a single factor or a single mechanism can cause many changes associated with this neuropathological phenomenon. MALDI mass spectrometry imaging (MSI) coupled to protein identification, because of its ability to study a wide range of molecules, appears to be suitable for the preparation of molecular profiles in TLE. Seven neuropeptides have been have been identified in Dental gyrus regions of the hippocampus in relation with TLE pathology. Shot-gun studies taking into account gender influence have been performed. Tissue microextraction from control (10) toward 10 TLE patients have been analyzed after trypsin digestion followed by separation on nanoLC coupled to LTQ orbitrap. From the shot-gun analyses, results confirmed the presence of specific neuropeptides precursors and receptors in TLE patients as well as proteins involved in axons regeneration including neurotrophins, ECM proteins, cell surface proteins, membrane proteins, G-proteins, cytoskeleton proteins and tumor suppressors. Among the tumor suppressors identified, the Leucine-rich glioma inactivated 1 (LGI1) protein was found. LGI1 gene recently been demonstrated being implicated in heritability of TLE. We have also demonstrate the presence a complete profile of tumor suppressors in TLE patients, 7 have been identified. Refining this analysis taken into account the gender influence in both control and in TLE reflected the presence of specific proteins between male and female and thus mechanisms in pathology development could be completely different.

Ionic matrices pre-spotted matrix-assisted laser desorption/ionization plates for patient maker following in course of treatment, drug titration, and MALDI mass spectrometry imaging.

In the current study, we compared plastic matrix-assisted laser desorption/ionization (MALDI) plates pre-spotted with different solid ionic matrices. Data reflect that after 3 months of storage, the standards were oxidized in α-cyano-4-hydroxycinnamic acid (HCCA) whether or not in HCCA/3-acetylpyridine (3APY) and HCCA/aniline, and certain peptides, such as ubiquitin, were not detected using the HCCA matrix, whereas they were detected in pre-spotted ionic matrices. Application in peptidomics of these MALDI matrices pre-spotted plates (after 3 months of storage) with ovarian cyst fluid showed less intense signals with HCCA than with solid ionic matrices. We show that these pre-spotted ionic matrices plates can be used for relative drug quantification, high mass protein detection, and MALDI mass spectrometry imaging.

The Antibody Dependant Neurite Outgrowth Modulation Response Involvement in Spinal Cord Injury.

Spinal cord injury (SCI) represents a major medical challenge. At present, there is still no cure to treat it efficiently and enable functional recovery below the injury site. Previously, we demonstrated that inflammation determines the fate of the physiopathology. To decipher the molecular mechanisms involved in this process, we performed a meta-analysis of our spatio-temporal proteomic studies in the time course of SCI. This highlighted the presence of IgG isotypes in both spinal cord explants and their secretomes. These IgGs were detected in the spinal cord even if no SCI occurred. However, during the time course following SCI, abundance of IgG1 and IgG2 subclasses (a, b, c) varied according to the spatial repartition. IgG1 was clearly mostly abundant at 12 h, and a switch to IgG2a was observed after 24 h. This IgG stayed predominant 3, 7, and 10 days after SCI. A protein related to IgM as well as a variable heavy chain were only detected 12 h after lesion. Interestingly, treatment with RhoA inhibitor influenced the abundance of the various IgG isotypes and a preferential switch to IgG2c was observed. By data reuse of rat dorsal root ganglion (DRG) neurons RNAseq datasets and RT-PCR experiments performed on cDNA from DRG sensory neurons ND7/23 and N27 dopaminergic neural cell lines, we confirmed expression of immunoglobulin heavy and light chains (constant and variable) encoding genes in neurons. We then identified CD16 and CD32b as their specific receptors in sensory neuron cell line ND7/23 and their activation regulated neurites outgrowth. These results suggest that during SCI, neuronal IgG isotypes are released to modulate neurites outgrowth. Therefore, we propose a new view of the SCI response involving an antibody dependent neurite outgrowth modulation (ADNM) which could be a precursor to the neuroinflammatory response in pathological conditions.

Caffeine intake exerts dual genome-wide effects on hippocampal metabolism and learning-dependent transcription.

Caffeine is the most widely consumed psychoactive substance in the world. Strikingly, the molecular pathways engaged by its regular consumption remain unclear. We herein addressed the mechanisms associated with habitual (chronic) caffeine consumption in the mouse hippocampus using untargeted orthogonal omics techniques. Our results revealed that chronic caffeine exerts concerted pleiotropic effects in the hippocampus at the epigenomic, proteomic, and metabolomic levels. Caffeine lowered metabolism-related processes (e.g., at the level of metabolomics and gene expression) in bulk tissue, while it induced neuron-specific epigenetic changes at synaptic transmission/plasticity-related genes and increased experience-driven transcriptional activity. Altogether, these findings suggest that regular caffeine intake improves the signal-to-noise ratio during information encoding, in part through fine-tuning of metabolic genes, while boosting the salience of information processing during learning in neuronal circuits.

AMASS: algorithm for MSI analysis by semi-supervised segmentation.

Mass Spectrometric Imaging (MSI) is a molecular imaging technique that allows the generation of 2D ion density maps for a large complement of the active molecules present in cells and sectioned tissues. Automatic segmentation of such maps according to patterns of co-expression of individual molecules can be used for discovery of novel molecular signatures (molecules that are specifically expressed in particular spatial regions). However, current segmentation techniques are biased toward the discovery of higher abundance molecules and large segments; they allow limited opportunity for user interaction, and validation is usually performed by similarity to known anatomical features. We describe here a novel method, AMASS (Algorithm for MSI Analysis by Semi-supervised Segmentation). AMASS relies on the discriminating power of a molecular signal instead of its intensity as a key feature, uses an internal consistency measure for validation, and allows significant user interaction and supervision as options. An automated segmentation of entire leech embryo data images resulted in segmentation domains congruent with many known organs, including heart, CNS ganglia, nephridia, nephridiopores, and lateral and ventral regions, each with a distinct molecular signature. Likewise, segmentation of a rat brain MSI slice data set yielded known brain features and provided interesting examples of co-expression between distinct brain regions. AMASS represents a new approach for the discovery of peptide masses with distinct spatial features of expression. Software source code and installation and usage guide are available at http://bix.ucsd.edu/AMASS/ .

Automated querying and identification of novel peptides using MALDI mass spectrometric imaging.

MSI is a molecular imaging technique that allows for the generation of topographic 2D maps for various endogenous and some exogenous molecules without prior specification of the molecule. In this paper, we start with the premise that a region of interest (ROI) is given to us based on preselected morphological criteria. Given an ROI, we develop a pipeline, first to determine mass values with distinct expression signatures, localized to the ROI, and second to identify the peptides corresponding to these mass values. To identify spatially differentiated masses, we implement a statistic that allows us to estimate, for each spectral peak, the probability that it is over- or under-expressed within the ROI versus outside. To identify peptides corresponding to these masses, we apply LC-MS/MS to fragment endogenous (nonprotease digested) peptides. A novel pipeline based on constructing sequence tags de novo from both original and decharged spectra and a subsequent database search is used to identify peptides. As the MSI signal and the identified peptide are only related by a single mass value, we isolate the corresponding transcript and perform a second validation via in situ hybridization of the transcript. We tested our approach, MSI-Query, on a number of ROIs in the medicinal leech, Hirudo medicinalis, including the central nervous system (CNS). The Hirudo CNS is capable of regenerating itself after injury, thus forming an important model system for neuropeptide identification. The pipeline helps identify a number of novel peptides. Specifically, we identify a gene that we name HmIF4, which is a member of the intermediate filament family involved in neural development and a second novel, uncharacterized peptide. A third peptide, derived from the histone H2B, is also identified, in agreement with the previously suggested role of histone H2B in axon targeting.

Mechanisms of innate events during skin reaction following intradermal injection of seasonal influenza vaccine.

The skin plays a crucial role in host defences against microbial attack and the innate cells must provide the immune system with sufficient information to organize these defences. This unique feature makes the skin a promising site for vaccine administration. Although cellular innate immune events during vaccination have been widely studied, initial events remain poorly understood. Our aim is to determine molecular biomarkers of skin innate reaction after intradermal (i.d.) immunization. Using an ex vivo human explant model from healthy donors, we investigated by NanoLC-MS/MS analysis and MALDI-MSI imaging, to detect innate molecular events (lipids, metabolites, proteins) few hours after i.d. administration of seasonal trivalent influenza vaccine (TIV). This multimodel approach allowed to identify early molecules differentially expressed in dermal and epidermal layers at 4 and 18 h after TIV immunization compared with control PBS. In the dermis, the most relevant network of proteins upregulated were related to cell-to-cell signalling and cell trafficking. The molecular signatures detected were associated with chemokines such as CXCL8, a chemoattractant of neutrophils. In the epidermis, the most relevant networks were associated with activation of antigen-presenting cells and related to CXCL10. Our study proposes a novel step-forward approach to identify biomarkers of skin innate reaction. SIGNIFICANCE: To our knowledge, there is no study analyzing innate molecular reaction to vaccines at the site of skin immunization. What is known on skin reaction is based on macroscopic (erythema, redness…), microscopic (epidermal and dermal tissues) and cellular events (inflammatory cell infiltrate). Therefore, we propose a multimodal approach to analyze molecular events at the site of vaccine injection on skin tissue. We identified early molecular networks involved biological functions such cell migration, cell-to-cell interaction and antigen presentation, validated by chemokine expression, in the epidermis and dermis, then could be used as early indicator of success in immunization.

Neuronal Activation in the Periaqueductal Gray Matter Upon Electrical Stimulation of the Bladder.

Reflexes, that involve the spinobulbospinal pathway control both storage and voiding of urine. The periaqueductal gray matter (PAG), a pontine structure is part of the micturition pathway. Alteration in this pathway could lead to micturition disorders and urinary incontinence, such as the overactive bladder symptom complex (OABS). Although different therapeutic options exist for the management of OABS, these are either not effective in all patients. Part of the pathology of OABS is faulty sensory signaling about the filling status of the urinary bladder, which results in aberrant efferent signaling leading to overt detrusor contractions and the sensation of urgency and frequent voiding. In order to identify novel targets for therapy (i.e., structures in the central nervous system) and explore novel treatment modalities such as neuromodulation, we aimed at investigating which areas in the central nervous system are functionally activated upon sensory afferent stimulation of the bladder. Hence, we designed a robust protocol with multiple readout parameters including immunohistological and behavioral parameters during electrical stimulation of the rat urinary bladder. Bladder stimulation induced by electrical stimulation, below the voiding threshold, influences neural activity in: (1) the caudal ventrolateral PAG, close to the aqueduct; (2) the pontine micturition center and locus coeruleus; and (3) the superficial layers of the dorsal horn, sacral parasympathetic nucleus and central canal region of the spinal cord. In stimulated animals, a higher voiding frequency was observed but was not accompanied by increase in anxiety level and locomotor deficits. Taken together, this work establishes a critical role for the vlPAG in the processing of sensory information from the urinary bladder and urges future studies to investigate the potential of neuromodulatory approaches for urological diseases.

The Adenosinergic Signaling: A Complex but Promising Therapeutic Target for Alzheimer's Disease.

Alzheimer's disease (AD) is the most common neurodegenerative disorder in elderly people. AD is characterized by a progressive cognitive decline and it is neuropathologically defined by two hallmarks: extracellular deposits of aggregated ß-amyloid (Aß) peptides and intraneuronal fibrillar aggregates of hyper- and abnormally phosphorylated Tau proteins. AD results from multiple genetic and environmental risk factors. Epidemiological studies reported beneficial effects of caffeine, a non-selective adenosine receptors antagonist. In the present review, we discuss the impact of caffeine and of adenosinergic system modulation on AD, in terms of pathology and therapeutics.

3D MALDI mass spectrometry imaging reveals specific localization of long-chain acylcarnitines within a 10-day time window of spinal cord injury.

We report, for the first time, the detection and specific localization of long-chain acylcarnitines (LC ACs) along the lesion margins in an experimental model of spinal cord injury (SCI) using 3D mass spectrometry imaging (MSI). Acylcarnitines palmitoylcarnitine (AC(16:0)), palmitoleoylcarnitine (AC(16:1)), elaidic carnitine (AC(18:1)) and tetradecanoylcarnitine (AC(14:1)) were detected as early as 3 days post injury, and were present along the lesion margins 7 and 10 days after SCI induced by balloon compression technique in the rat. 3D MSI revealed the heterogeneous distribution of these lipids across the injured spinal cord, appearing well-defined at the lesion margins rostral to the lesion center, and becoming widespread and less confined to the margins at the region located caudally. The assigned acylcarnitines co-localize with resident microglia/macrophages detected along the lesion margins by immunofluorescence. Given the reported pro-inflammatory role of these acylcarnitines, their specific spatial localization along the lesion margin could hint at their potential pathophysiological roles in the progression of SCI.

Behavioral effects of deep brain stimulation of different areas of the Papez circuit on memory- and anxiety-related functions.

Deep brain stimulation (DBS) has gained interest as a potential therapy for advanced treatment-resistant dementia. However, possible targets for DBS and the optimal stimulation parameters are not yet clear. Here, we compared the effects of DBS of the CA1 sub-region of the hippocampus, mammillothalamic tract, anterior thalamic nucleus, and entorhinal cortex in an experimental rat model of dementia. Rats with scopolamine-induced amnesia were assessed in the object location task with different DBS parameters. Moreover, anxiety-related side effects were evaluated in the elevated zero maze and open field. After sacrifice, we applied c-Fos immunohistochemistry to assess which memory-related regions were affected by DBS. When comparing all structures, DBS of the entorhinal cortex and CA1 sub-region was able to restore memory loss when a specific set of stimulation parameters was used. No anxiety-related side effects were found following DBS. The beneficial behavioral performance of CA1 DBS rats was accompanied with an activation of cells in the anterior cingulate gyrus. Therefore, we conclude that acute CA1 DBS restores memory loss possibly through improved attentional and cognitive processes in the limbic cortex.

Multiple changes in peptide and lipid expression associated with regeneration in the nervous system of the medicinal leech.

BACKGROUND: The adult medicinal leech central nervous system (CNS) is capable of regenerating specific synaptic circuitry after a mechanical lesion, displaying evidence of anatomical repair within a few days and functional recovery within a few weeks. In the present work, spatiotemporal changes in molecular distributions during this phenomenon are explored. Moreover, the hypothesis that neural regeneration involves some molecular factors initially employed during embryonic neural development is tested. RESULTS: Imaging mass spectrometry coupled to peptidomic and lipidomic methodologies allowed the selection of molecules whose spatiotemporal pattern of expression was of potential interest. The identification of peptides was aided by comparing MS/MS spectra obtained for the peptidome extracted from embryonic and adult tissues to leech transcriptome and genome databases. Through the parallel use of a classical lipidomic approach and secondary ion mass spectrometry, specific lipids, including cannabinoids, gangliosides and several other types, were detected in adult ganglia following mechanical damage to connected nerves. These observations motivated a search for possible effects of cannabinoids on neurite outgrowth. Exposing nervous tissues to Transient Receptor Potential Vanilloid (TRPV) receptor agonists resulted in enhanced neurite outgrowth from a cut nerve, while exposure to antagonists blocked such outgrowth. CONCLUSION: The experiments on the regenerating adult leech CNS reported here provide direct evidence of increased titers of proteins that are thought to play important roles in early stages of neural development. Our data further suggest that endocannabinoids also play key roles in CNS regeneration, mediated through the activation of leech TRPVs, as a thorough search of leech genome databases failed to reveal any leech orthologs of the mammalian cannabinoid receptors but revealed putative TRPVs. In sum, our observations identify a number of lipids and proteins that may contribute to different aspects of the complex phenomenon of leech nerve regeneration, establishing an important base for future functional assays.

Liquid ionic matrixes for MALDI mass spectrometry imaging of lipids.

Lipids are a major component of cells and play a variety of roles in organisms. In general, they play a key role in the structural composition of membranes. Some lipids, such as sphingoglycolipids, however, are also mediators of different biological processes, including protein transport, regulation of cell growth, cellular morphogenesis, neuronal plasticity, and regulation of the immune response. With the advent of MALDI mass spectrometry imaging (MALDI MSI), lipids have begun to be intensively investigated by several groups. Here we present a novel development in the detection and study of lipids using an automatic microspotter coupled to specific liquid ionic matrixes based on a 2,5-DHB matrix (i.e., 2,5-DHB/ANI, 2,5-DHB/Pyr, and 2,5-DHB/3-AP). This development allows to decrease the time of the sample preparation by comparison with crystalline 2,5-DHB as matrix and was validated on human ovarian cancer biopsies to demonstrate its use as a precise procedure that is particularly useful for specific diagnoses.

Localization of secondary metabolites in marine invertebrates: contribution of MALDI MSI for the study of saponins in Cuvierian tubules of H. forskali.

BACKGROUND: Several species of sea cucumbers of the family Holothuriidae possess a particular mechanical defense system called the Cuvierian tubules (Ct). It is also a chemical defense system as triterpene glycosides (saponins) appear to be particularly concentrated in Ct. In the present study, the precise localization of saponins in the Ct of Holothuria forskali is investigated. Classical histochemical labeling using lectin was firstly performed but did not generate any conclusive results. Thus, MALDI mass spectrometry Imaging (MALDI-MSI) was directly applied and completed by statistical multivariate tests. A comparison between the tubules of relaxed and stressed animals was realized. RESULTS: These analyses allowed the detection of three groups of ions, corresponding to the isomeric saponins of the tubules. Saponins detected at m/z 1287 and 1303 were the most abundant and were apparently localized in the connective tissue of the tubules of both relaxed and stressed individuals. Saponins at m/z 1125 and 1141 were detected in lower amount and were present in tissues of relaxed animals. Finally, saponin ions at 1433, 1449, 1463 and 1479 were observed in some Ct of stressed holothuroids in the outer part of the connective tissue. The saponin group m/z 14xx seems therefore to be stress-specific and could originate from modifications of the saponins with m/z of 11xx. CONCLUSIONS: All the results taken together indicate a complex chemical defense mechanism with, for a single organ, different sets of saponins originating from different cell populations and presenting different responses to stress. The present study also reflects that MALDI-MSI is a valuable tool for chemical ecology studies in which specific chemical signalling molecules like allelochemicals or pheromones have to be tracked. This report represents one of the very first studies using these tools to provide a functional and ecological understanding of the role of natural products from marine invertebrates.