B Berthon

M Evans

C Marshall

N Palaniappan

N Cole

V Jayaprakasam

T Rackley

E Spezi

Dr Berthon

Head and neck target delineation using a novel PET automatic segmentation algorithm

Keywords:

Purpose: To evaluate the feasibility and impact of using a novel advanced PET auto-segmentation method in Head and Neck (H&N) radiotherapy treatment (RT) planning. Methods: ATLAAS, Automatic decision Tree-based Learning Algorithm for Advanced Segmentation, previously developed and validated on pre-clinical data, was applied to 18 F-FDG-PET/CT scans of 20 H&N patients undergoing Intensity Modulated Radiation Therapy. Primary Gross Tumour Volumes (GTVs) manually delineated on CT/MRI scans (GTVp CT/MRI ), together with ATLAAS-generated contours (GTVp ATLAAS ) were used to derive the RT planning GTV (GTVp final ). ATLAAS outlines were compared to CT/MRI and final GTVs qualitatively and quantitatively using a conformity metric. Results: The ATLAAS contours were found to be reliable and useful. The volume of GTVp ATLAAS was smaller than GTVp CT/MRI in 70% of the cases, with an average conformity index of 0.70. The information provided by ATLAAS was used to grow the GTVp CT/MRI in 10 cases (up to 10.6 mL) and to shrink the GTVp CT/MRI in 7 cases (up to 12.3 mL). ATLAAS provided complementary information to CT/MRI and GTVp ATLAAS contributed to up to 33% of the final GTV volume across the patient cohort. Conclusions: ATLAAS can deliver operator independent PET segmentation to augment clinical outlining using CT and MRI and could have utility in future clinical studies.

Positron Emission Tomography (PET) imaging using 18F-Fluorodeoxyglucose (FDG) plays an increasingly valuable role in Radiotherapy Treatment (RT) planning for a number of cancers [START_REF] Macmanus | Use of PET and PET/CT for radiation therapy planning: IAEA expert report 2006-2007[END_REF]. Loco-regional recurrences have been shown to occur in PETavid regions of the tumour [START_REF] Due | Recurrences after intensity modulated radiotherapy for head and neck squamous cell carcinoma more likely to originate from regions with high baseline [18F]-FDG uptake[END_REF] and studies have demonstrated the feasibility and usefulness of PET/CT-guided Intensity Modulated Radiation Therapy (IMRT) planning [START_REF] Wang | Initial experience of FDG-PET/CT guided IMRT of Head and Neck carcinoma[END_REF]. PET/CT-based outlining can lead to more accurate and reproducible delineation of the Gross Tumour Volume (GTV), compared to outlining done using CT alone [START_REF] Daisne | Tumor volume in pharyngolaryngeal squamous cell carcinoma: comparison at CT, MR imaging, and FDG PET and validation with surgical specimen[END_REF]. The PET-based GTV is usually smaller than the CT based volume [START_REF] Ciernik | Radiation treatment planning with an integrated positron emission and computer tomography (PET/CT): a feasibility study[END_REF][START_REF] Deantonio | FDG-PET/ CT imaging for staging and radiotherapy treatment planning of head and neck carcinoma[END_REF]. Nishioka et al. showed with 21 oropharyngeal and nasopharyngeal cancer patients that adjacent normal tissue, particularly parotids, could be spared in 71% of patients when using PET in the delineation process [START_REF] Nishioka | Image fusion between 18 FDG-PET and MRI/CT for radiotherapy planning of oropharyngeal and nasopharyngeal carcinomas[END_REF], which could potentially lead to reduced long term morbidity, xerostomia and improved quality of life.

Although FDG-PET has been adopted in oncology as a key tool in diagnostic imaging, its use in RT planning has, until now, been limited due to a lack of consensus on GTV delineation method. The low resolution of PET coupled with the proximity to the tumour of other metabolically active structures make the delineation challenging. In particular in the Head and Neck (H&N), organs such as the pharyngeal muscles, spinal cord and salivary glands, which should be spared to minimise morbidity and improve quality of life, can generate additional background FDG uptake.

Manual PET-based GTV delineation, currently used in most centres, is time consuming and highly operator-dependent and several studies have shown significant variations in the GTV delineated by different operators using PET [START_REF] Ciernik | Radiation treatment planning with an integrated positron emission and computer tomography (PET/CT): a feasibility study[END_REF][START_REF] Caldwell | Observer variation in contouring gross tumor volume in patients with poorly defined non-small-cell lung tumors on CT: the impact of 18FDG-hybrid PET fusion[END_REF]. This has led to the development and recommended use of various PET automatic segmentation (PET-AS) methods for H&N [START_REF] Grégoire | Radiotherapy for head and neck tumours in 2012 and beyond: conformal, tailored, and adaptive?[END_REF]. However, only a small number of prospective clinical studies have reported on the use of PET-AS in RT planning [START_REF] Deantonio | FDG-PET/ CT imaging for staging and radiotherapy treatment planning of head and neck carcinoma[END_REF]. Comparing different studies is difficult because of the different PET-AS methods used. Basic thresholding methods lack accuracy and reliability [START_REF] Nestle | Comparison of different methods for delineation of 18F-FDG PET-positive tissue for target volume definition in radiotherapy of patients with non-small cell lung cancer[END_REF][START_REF] Ford | Tumor delineation using PET in head and neck cancers: threshold contouring and lesion volumes[END_REF], but more advanced PET-AS methods, such as gradient-based, clustering or region-growing approaches are rarely used, and their impact on RT planning is still unclear. There is a need for studies investigating the feasibility and clinical benefits of using advanced PET-AS in RT planning.

This prospective study investigated the use of an optimised PET-AS tool, developed and validated in house using phantom and clinical PET data [START_REF] Berthon | Influence of cold walls on PET image quantification and volume segmentation[END_REF][START_REF] Berthon | Evaluation of advanced automatic PET segmentation methods using non-spherical thin-wall inserts[END_REF], for GTV delineation in the RT planning of 20 oropharyngeal cancer patients. We evaluated the feasibility and impact of including this method into the RT planning process.

Methods

The ATLAAS optimised segmentation model PET-AS was performed using the Automatic decision Tree-based Learning Algorithm for Advanced Segmentation (ATLAAS)2 method developed at our centre. The ATLAAS model is designed to select the most accurate PET-AS method for a given PET image. This is achieved using a decision tree supervised machine learning method, optimised with a training dataset for which the segmentation outcome is known, to achieve optimal performance for cases in which the outcome is not known. ATLAAS is described elsewhere [START_REF] Berthon | ATLAAS: an Automatic decision treebased learning algorithm for advanced segmentation[END_REF], and its accuracy was shown for 6 classes of advanced PET-AS methods used to segment a large range of data including simulated H&N tumours, and phantom H&N images of complex and realistic tumours obtained with a sub-resolution printed sandwich phantom [START_REF] Berthon | A novel phantom technique for evaluating the performance of PET auto-segmentation methods in delineating heterogeneous and irregular lesions[END_REF]. ATLAAS was optimised for H&N data using 65 sub-resolution printed sandwich phantom images. The optimised version included the two algorithms Adaptive Thresholding method (AT) and Gaussian mixture models Clustering Method using 5 clusters (GCM5), described in previous work [START_REF] Berthon | Influence of cold walls on PET image quantification and volume segmentation[END_REF]. The best method was predicted on the basis of TBR peak defined as the ratio between the tumour peak intensity value, (mean value in a 1 cm 3 sphere centred on the maximum intensity voxel) and the background intensity (mean intensity in a 1 cm thick extension of a thresholded volume at 50% of the peak intensity value). An example of the typical steps involved in the segmentation with ATLAAS is given in Fig. 1. The ATLAAS model was implemented for this work in the Computational Environment for Radiotherapy Research (CERR) [START_REF] Deasy | CERR: a computational environment for radiotherapy research[END_REF]. The segmentation accuracy was evaluated by quantifying the overlap between the segmented and true contour using the Dice Similarity Coefficient (DSC) described in other work [START_REF] Dice | Measures of the amount of ecologic association between species[END_REF].

Acquisition of clinical data

The POSITIVE (Optimization of Positron Emission Tomography based Target Volume Delineation in Head and Neck Radiotherapy) study was set up to test ATLAAS for the first time in patients under-going H&N radiotherapy (REC No. 12/WA/0083) and was carried out at Velindre Cancer Centre (UK). Twenty stage III/IVa-b oropharyngeal cancer patients were recruited after informed consent to the study. The patients were treated with neoadjuvant (induction) chemotherapy followed by radical chemoradiotherapy (66 Gy in 30 fractions over 6 weeks) using IMRT. A planning FDG PET/CT scan was carried out on a GE Discovery 690 PET/CT scanner before chemotherapy to avoid changes in tumour volumes prior to outlining. The scans were acquired 90 min after FDG administration in the treatment position with an RT immobilisation shell. The PET was acquired using 6-8 bed positions of 3 min each. The patient was injected with contrast for a subsequent CT used in the planning process. The images were reconstructed to 512 Â 512 voxels for CT and 256 Â 256 voxels for PET, using the algorithm Vue Point FX (24 subsets, 2 iterations, 6.4 mm cut-off) including CT-based attenuation-, scatter-and Time-Of-Flight corrections.

Six weeks on average separated the PET/CT planning scan and the start of RT. The fit of the immobilisation shells was adjusted if needed after induction chemotherapy and the patient was reoutlined and re-planned if necessary using the original CT/MRI/ PET scan. Reporting was done by PET specialist radiologists after acquisition of the planning scans.

Workflow and analysis

MRI scans acquired before recruitment were available for all patients and were fused to the planning PET-CT scan using the Mutual Information registration algorithm in the ProSoma software (MedCom GmbG, Darmstadt, Germany).

Planning scans for the first 10 patients recruited were used to validate the workflow and verify that ATLAAS provided relevant contours for use. In this subgroup the primary GTVs were manually outlined by three consultant radiation oncologists, in discussion with a specialist PET radiologist, on the registered PET/CT, using the software VelocityAI (Varian Medical Systems, Palo Alto, USA). The resulting GTVp PET/CT contours were compared with ATLAAS contours in terms of their volume and geometrical overlap, using the DSC index.

Once the ATLAAS output was verified, another 10 oropharyngeal cancer patients were recruited for the study. Manual delineation of the primary GTV was performed by the consultant radiation oncologists on the fused MRI and CT images in ProSoma (GTVp CT/MRI ). ATLAAS (GTVp ATLAAS ) contours were then imported into ProSoma where the final GTV (GTVp final ) was drawn by the treating clinician using all the available contour data.

The use of the additional information brought by ATLAAS contours was evaluated by comparing the different contours (GTVp CT/MRI , GTVp ATLAAS , and GTVp final ) for each patient, in terms of volume and geometrical overlap using the DSC. In addition, the clinicians were asked to report any changes made to the GTVp final due to the ATLAAS contour. Lymph nodes, which are well defined on CT/MRI images, were not outlined using ATLAAS, and are therefore not reported on in this paper.

Results

The patient cohort included 17 men and 3 women with a median age of 63 years. Ten patients had tonsillar tumours, 8 base of tongue tumours and 2 soft palate tumours. Two patients needed re-planning after induction chemotherapy.

In the preliminary group of 10 patients, ATLAAS successfully delineated the PET-avid tumour for all patients. The segmentation of the tumour ROI was fully automatic and took no more than 2 min on a dual core 3.1 GHz processor. GTVp ATLAAS were smaller than the manually delineated GTVp PET/CT for 7 out of 10 patients. The mean DSC between GTVp PET/CT and GTVp ATLAAS was 0.82, when 0.7 is considered to be an indicator of good overlap [START_REF] Zou | Statistical validation of image segmentation quality based on a spatial overlap index[END_REF]. On the basis of these results, it was decided that only ATLAAS and CT/MRI contours would be used for the subsequent 10 patients recruited.

A comparison in terms of volume and conformity between the GTVp delineated using ATLAAS and both CT/MRI-based and final contours delineated by the investigators is presented in Table 1. ATLAAS volumes were smaller than the corresponding CT/MRI volumes in 7 out of 10 cases, and were within 10% of CT/MRI volumes in 4 out of 10 cases. The spatial conformity of GTVp ATLAAS and GTVp CT/MRI was 0.70 DSC on average. GTVp ATLAAS and GTVp final were close, with neither being larger than 30% of the other, in 6 out of the 10 cases. GTVp final volumes were larger than the GTVp ATLAAS in all cases. However, the ATLAAS volumes showed good conformity to the final contour, with an average DSC of 0.77. Fig. 2 reports the details of the global and local changes to the final volume based on ATLAAS, and outlines the differences between ATLAAS and CT/MRI contours not taken into account in the final GTV. For instance, the data in the top row of the table shows that more than 83% of the ATLAAS volume was included in the final GTV for all patients, and 100% of the ATLAAS volume was included in the final GTV in 4 cases. The second row reports the proportion of the CT/MRI volume modified on the basis of the ATLAAS outline. This value ranged from 6.5% to 33%. This modification could include both additional extension of the volume when the ATLAAS contour was outside the GTVp CT/MRI or local reduction of the volume in cases where the inverse was true. This is detailed in rows 3-5 as illustrated under the table.

Fig. 3 illustrates specific differences found between GTVp CT/MRI , GTVp ATLAAS and GTVp final overlaid on the corresponding CT/PET scan, for seven clinical cases of interest.

Extending The GTV locally based on ATLAAS

As reported in the third row of Fig. 2, GTVp CT/MRI was locally extended based on the information provided by ATLAAS (cf. Fig. 3a) for all clinical cases, with up to 10 mL added to make the final volume. Visual examination and reporting by the clinicians showed that this was done when additional disease extension was detected by ATLAAS, and confirmed by clinical or CT/MRI findings. This included larger superior-inferior disease extension (by up to 1.1 cm in one patient as reported in Fig. 3b), and disease extension identified across the midline (cf. Fig. 3c).

Reducing the GTV locally based on ATLAAS

As shown in the fourth row of Fig. 2, local reduction (on one or more transverse slices) of the CT/MRI volume based on ATLAAS was observed for 7 patients. For two patients, it resulted in a reduction to the final volume of more than 2 mL. The extent of the contours was locally reduced when the smaller disease extent indicated by ATLAAS was in agreement with the clinical findings and the CT or the MRI information. The extent was also reduced in the superior-inferior direction for two patients (by 1.5 cm for patient [START_REF] Deasy | CERR: a computational environment for radiotherapy research[END_REF]. In cases of largely conflicting information between image modalities, the CT/MRI contour was reduced down to a compromise following the edge of the anatomical structures, as depicted in Fig. 3d.

ATLAAS information discarded

Differences between GTVp CT/MRI and GTVp ATLAAS were not considered in the final GTV when they included: (a) bone (0.1 mL for patient No 11, cf. Fig. 3e), (b) air (for 5 patients, up to 6.6 mL for patient No 12, cf. Fig. 3f), (c) different superior-inferior disease extension in GTVp ATLAAS which was not confirmed by anatomical imaging or clinical examinations (for 6 patients, up to 6.4 mL for patient No 13, cf. Fig. 3g), (d) different transverse disease extension unconfirmed by anatomical imaging or clinical examinations (cf. some regions in Fig. 3f).

In these cases, the difference between GTVp ATLAAS and GTVp final (expressed in mL), is given in row 5 of Fig. 2 and includes both over and under contouring.

Discussion

In this study, we investigated the clinical feasibility of using the novel ATLAAS optimised segmentation model in 20 H&N cancer patients undergoing radical chemoradiotherapy. ATLAAS was applied for the first time to 20 prospectively recruited patients in a clinical trial with a strict scanning protocol, which involved expert PET radiologist and H&N radiation oncologists. It was prospectively used, in combination with manual CT/MRI data, to derive the final GTV for use in RT planning. To the best of our knowledge, advanced PET-AS methods (beyond simple thresholding) have only been included as part of RT treatment planning in two studies in H&N cancer [START_REF] Geets | Adaptive biological image-guided IMRT with anatomic and functional imaging in pharyngolaryngeal tumors: impact on target volume delineation and dose distribution using helical tomotherapy[END_REF][START_REF] Leclerc | Primary tumor delineation based on 18FDG PET for locally advanced head and neck cancer treated by chemo-radiotherapy[END_REF], which were based on the same gradient-based segmentation method. In this work, we additionally evaluated the impact of using the PET-AS contour on local modifications of the final GTV contour.

ATLAAS had previously shown accuracy and robustness on phantom and simulated data for the evaluation of H&N PET scans [START_REF] Berthon | ATLAAS: an Automatic decision treebased learning algorithm for advanced segmentation[END_REF]. Evaluation on images from the 10 first patients involved in this study showed that ATLAAS provided PET-avid GTVs for all patients with a high degree of similarity to PET GTVs manually delineated by experts. In addition, the segmentation was fully automatic and therefore reproducible, and lasted no more than 2 min per patient. The use of ATLAAS instead of manual PET/CT outlining for the 10 subsequent patients in this study, considerably reduced the clinicians' workload and removed inter-observer variability. We have shown that ATLAAS not only could segment the PET-avid areas of disease reliably in patients compared to manual PET outlining but that it could also add valuable information to guide clinical delineation of the primary GTV.

The ATLAAS contours were smaller than the CT/MRI contours in most cases, which is in agreement with findings from other studies where threshold-based delineation was used for H&N patients [START_REF] Greco | Evaluation of different methods of 18F-FDG-PET target volume delineation in the radiotherapy of head and neck cancer[END_REF]. Furthermore, the ATLAAS derived contours provided additional information to anatomical contours manually drawn on CT and MRI. This is in line with the study by Newbold et al. in 19 H&N patients, where threshold-based delineation was used to derive the PET-based GTV [START_REF] Newbold | Evaluation of the role of 18FDG-PET/CT in radiotherapy target definition in patients with head and neck cancer[END_REF]. In our study, we found that additional information from ATLAAS included (a) identification of superior-inferior disease extension, and extension across the midline not seen on CT (e.g. Fig. 3c), and (b) other disease extension boundaries differing from anatomical data. The information provided by ATLAAS was used in all patients and this shows the confidence of the clinicians in the usefulness of our segmentation method for RT planning at our centre. The clinician's judgment and expertise and the additional clinical data available (endoscopy or clinical examination results) remained paramount in the process. Nevertheless ATLAAS was very useful (a) in confirming the GTV outline when this was close to the CT/MRI based contour, and (b) as a delineation guide when in disagreement with CT/ MRI based contours, due for instance to different patient positioning and/or poor image registration.

We have methodically investigated the impact of ATLAAS on the final GTV for our cohort. We found that although ATLAAS led to reducing the extension in some areas of the GTVp CT/MRI for 7 patients, the PET information led to a globally smaller final GTV for only 1 patient. This is in line with the findings of Ciernik et al. for a cohort of 12 H&N patients [START_REF] Ciernik | Radiation treatment planning with an integrated positron emission and computer tomography (PET/CT): a feasibility study[END_REF], and Paulino et al. for 40 H&N patients [START_REF] Paulino | Comparison of CT-and FDG-PET-defined gross tumor volume in intensity-modulated radiotherapy for head-and-neck cancer[END_REF], both using manual PET segmentation. This confirms the suggestion that clinicians may not be prepared yet to reduce the GTV volume based on PET. Indeed, although some studies have shown that PET-AS contours can accurately identify the whole tumour burden in laryngeal cancer [START_REF] Daisne | Tumor volume in pharyngolaryngeal squamous cell carcinoma: comparison at CT, MR imaging, and FDG PET and validation with surgical specimen[END_REF][START_REF] Geets | A gradient-based method for segmenting FDG-PET images: methodology and validation[END_REF], it may be more useful for defining the metabolically active tumour region, especially for tumours which can be highly heterogeneous such as in the H&N [START_REF] Hoogsteen | Tumor microenvironment in head and neck squamous cell carcinomas: predictive value and clinical relevance of hypoxic markers. A review[END_REF]. This is in line with the suggestion of considering the Biological Tumor Volume as defined by Ling et al. [START_REF] Ling | Towards multidimensional radiotherapy (MD-CRT): biological imaging and biological conformality[END_REF], which can be used for dose escalation [START_REF] Van Elmpt | The PET-boost randomised phase II dose-escalation trial in nonsmall cell lung cancer[END_REF][START_REF] Even | PET-based dose painting in non-small cell lung cancer: comparing uniform dose escalation with boosting hypoxic and metabolically active sub-volumes[END_REF] to increase the dose to the biologically active tumour while sparing the surrounding tissue. The ATLAAS model could be useful for determining, with a consistent and operator independent approach, highly metabolically active areas of the tumour requiring a radiation boost and it could therefore be extremely useful for treatment plan adaptation. Correlation with additional information and clinical input would still be required in finalising the volumes for dose escalation.

Differences between the GTVp final and GTVp ATLAAS volumes were in the range [0.6,45] mL (cf. row 5 of Fig. 2). CT/MRI based outlining was preferred when: (a) no PET signal was found in abnormal mucosa (Fig. 3g), (b) high PET uptake was observed in and around air cavities and/or bone (Fig. 3c andh) due to signal spill-out or inflammation. Spill-out effects can be corrected with CT-based thresholding, whereas unconfirmed soft tissue extensions of the disease, which represent a large part of the differences observed between CT/MRI and PET contours (cf. rows 5c and 5d of Fig. 2), are inherent to the difference between modalities.

One of the limitations of this study is that we could not carry out a full comparison between GTVp ATLAAS and the PET GTV outlined manually without reference to anatomical data from the CT scan. In this case the correlation between manual and GTVp ATLAAS could have been greater because based on the same underlying data. In addition, this work was carried out as a single centre study. Both limitations shall be addressed in the design of a forthcoming multicentre clinical trial.

Conclusions

The ATLAAS optimised segmentation model based on the decision tree machine learning method is a novel, fast and operator independent tool for tumour delineation in radiotherapy treatment planning of Head and Neck cancer. ATLAAS can potentially be applied to any tumour site and tumour type and holds promise for future multi-centre clinical studies investigating the use of PET in radiotherapy outlining, prior to starting treatment and also for adaptive re-planning of residual metabolically active disease during treatment.

Fig. 1 .

 1 Fig. 1. Example of steps in the decision tree method implemented in the ATLAAS segmentation model.

Fig. 2 .

 2 Fig. 2. Quantification of the changes to the final volume (locally reduced or increased extent) based on the ATLAAS outlines, and differences between ATLAAS and CT/MRI not taken into account in the final GTV. Calculations corresponding to the different rows are schematically described under the table.

Fig. 3 .

 3 Fig. 3. GTV CT/MRI , GTVp ATLAAS , and GTVp final compared for 7 clinical cases.

Table 1

 1 GTVp volumes and DSC index for manual and ATLAAS contours.

	Patient no

Patent pending No PCT/GB2015/052981.

Please cite this article in press as: Berthon B et al. Head and neck target delineation using a novel PET automatic segmentation algorithm. Radiother Oncol (2017), http://dx.doi.org/10.1016/j.radonc.2016.12.008

Acknowledgements

This work was funded by Cancer Research Wales grants No. 7061 and 2476. The funder was not involved in the study design, conception, data collection acquisition and analysis nor writing or submission of this manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.


Armelle Garcia

Pamela Abboud

Emma Sylvestre

Fanny Jouniaux

Nicolas N Darcel

Suzanne Higgs

Olga Davidenko

Social modeling of low and high energy density starters and desserts

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Social modeling of low and high energy density starters and desserts

The British

Results

Context et Objective

• Social Modeling consits in using others' behavior as a guide for appropriate consumption.

• The majority of studies on social modeling were interested in food intakes.

• Much less is known about social modeling of food choices, when exposed to a various range of foods, including both high and low energy density items.

• A previous study conducted in real-conditions observed social modeling of food choices for starters but not for desserts, suggesting that all food categories are not similarly suceptible to social modeling (Garcia et al, 2021).

The aim of this study is to caracterize social modeling of food choice for different food categories (starters and desserts) and of both high and low energy density.

Methods

Protocol

Conclusion / Discussion

 We did not observe social modeling of low and high energy density choices.

-> Choices may be less suceptible to external influences compared to quantities because people may feel more certain of their food preferences than the adequate quantities to consumed, and thus do not look for guidance in their choices.

-> Low appreciation of high energy density items + Low number of items: reduced the uncertainty regarding preferences, and thus the suceptiblity to external influences.

-> Biases linked to the study setup? (ex. layout: participant and commensal ate back to back)

 Liking appeared as the main determinant of participants' food choices in the experiment.

 Energy intake was higher in session 1 compared to session 2, due to a higher consumption of high energy density desserts in session 1, which could be due to the novelty effect of the buffet.

Statistical Analysis

o Food choices distribution between "healthy" and "unhealthy" items according to the condition and the session : McNemar tests 40 volonteers (Women,normal BMI (18,5 et

  .garcia @agroparistech.fr

o

  Energy intake according to the condition and the session T-tests

  25 kg/m 2 ), no specific diet, no eating disorders)

Tomatoes cucumber

Carrots Celery

Sausage

Ham/Coppa

Lasagna

Lemon Pie

Fruits salad Plain Yogurt

Chocolate cake

Arrival of volunteers

•Waiting out of the room with the commensal • Cross words (5 min)

Entrance in the buffet room p < 0,01 p = 0,01 p < 0,01 p = 0,04 p = 0,66 p = 0,052 p = 0,01 p < 0,01 Expected satiety p = 0,67 p =0 ,74 p = 0,01 p = 0,39 p = 0,12 p = 0,30 p = 0,86 p = 0,40 Perceived Nutritional Quality p = 0,06 p = 0,08 p = 0,02 p = 0,43 p = 0,26 p = 0,97 p = 0,86 p = 0,09

 No significant difference in energy intake between « Healthy » and « Unhealthy » conditions.

 Significantly higher consumption of energy intake during session 1 compared to session 2  Energy intake difference between the sessions was due to a higher consumption of high energy density desserts in session 1

 No significant difference in the distribution of choices between low and high energy density items according to the condition or the session, neither for desserts nor for starters.

 No social modeling observed.

 Liking is the main factor predicting food choices SHIFT (ANR-18-CE21-0008)

Armelle Garcia 1 , Pamela Abboud 1 , Emma Sylvestre 1 , Fanny Jouniaux 1 , Nicolas Darcel 1 , Suzanne Higgs 2 , Olga Davidenko


Anne Roumier
email: anne.roumier@inserm.fr

Giulia Albertini
email: giulia.albertini@inserm.fr

Regulation of microglia by neuromodulators: modulations in major and minor modes

Keywords: Microglia, neuromodulators, inflammation, development, norepinephrine, serotonin, acetylcholine, histamine, dopamine, motility, chemotaxis, phagocytosis serotonin Aß: amyloid ß peptide ACh: acetylcholine ATP: adenosine triphosphate Ca 2+ : calcium cAMP: cyclic adenosine monophosphate CNS: central nervous system EGFP: enhanced green fluorescent protein GPCR

Microglial cells, the brain resident macrophages, participate to brain development and function and help maintaining its homeostasis. To play these roles, they need to detect and adapt to modifications of their environment, including changes in the activity of neurons. The neuromodulators serotonin, dopamine, norepinephrine, acetylcholine and histamine are synthesized and released by specialized neurons to coordinate the activity of other neurons in different regions. In this review, we summarize the current evidence obtained in vitro or in vivo that neuromodulators act on microglia. On the short term, they can modify their motility, morphology and phagocytic activity; on the mid-long term they can modulate their transition between different immune activation states. Lastly, we review some recent data suggesting that these regulations of microglia by neuromodulators are involved in vivo in some aspects of central nervous system development, function and homeostasis.

Introduction

Microglia are the resident tissue macrophages of the central nervous system (CNS). Besides their involvement in the immune response, they have been proposed as mediators of CNS development, contributors of CNS homeostasis and functions, and key players during neurodegenerative and psychiatric disorders. To engage in these roles, they need to detect and adapt to modifications of their environment, like the presence of pathogens or cell death, but also to global changes in neuronal activity. Neuronal activity in specific brain areas can be coordinated by specialized neurons which synthesize and release the so-called neuromodulators serotonin (5-HT), dopamine, norepinephrine, acetylcholine (ACh) and histamine. One intriguing hypothesis is that these typical neuromodulators1 might not only act on neuronal targets, but also directly tune microglia in harmony. Indeed, neuromodulators, which are mainly released through asynaptic volume transmission, can reach targets located a few micrometers away [1-5], and early ultrastructural studies identified glial elements close to sites of neuromodulators release [1,[START_REF] Ridet | Evidence for nonsynaptic serotonergic and noradrenergic innervation of the rat dorsal horn and possible involvement of neuron-glia interactions[END_REF][START_REF] Aoki | Beta-adrenergic receptors: astrocytic localization in the adult visual cortex and their relation to catecholamine axon terminals as revealed by electron microscopic immunocytochemistry[END_REF]. This review aims to provide the major keys to appreciate how neuromodulators can strengthen or inhibit microglial functions and properties. We will show, based on studies on primary cultures of microglia, brain slices or in vivo, that neuromodulators can act on the short term, by modulating microglia motility, morphology, phagocytic activity, and on the mid and long terms, by driving or modulating their transition between different immune activation states. Lastly, we will discuss some recent data suggesting that these regulations participate to CNS development, function and homeostasis.

1.

The regulation of microglia by neuromodulators is allowed by spatial vicinity between microglial processes and axons, and the expression of neuromodulator receptors. The ability of microglia to respond to a subset of neuromodulators, neurotransmitters and neuropeptides has been examined in systematic studies on microglia in primary culture or freshly purified from whole brain. Using intracellular calcium (Ca 2+ ) rise as a readout, these studies showed that a fraction (2 -20%) of microglial cells is responsive to 5-HT, dopamine, nicotine and histamine. These percentages are generally higher after a 24h-treatment with lipopolysaccharide (LPS), interferon (IFN)-γ or interleukin (IL)-4 [8,9]. Although the intensity of the elicited Ca 2+ rise and the percentage of responsive cells are lower than for adenosine triphosphate (ATP), the gold standard for Ca 2+ response in microglia, these studies demonstrate that microglia can respond to each of these neuromodulators. In addition, interestingly, different subpopulations respond either to one or to subsets of neuromodulators [9]. This functional diversity is consistent with recent global analyses documenting the heterogeneity of microglia among brain regions [START_REF] De Biase | Local Cues Establish and Maintain Region-Specific Phenotypes of Basal Ganglia Microglia[END_REF][START_REF] Grabert | Microglial brain region-dependent diversity and selective regional sensitivities to aging[END_REF][START_REF] Ayata | Epigenetic regulation of brain regionspecific microglia clearance activity[END_REF] and during development [START_REF] Li | Developmental Heterogeneity of Microglia and Brain Myeloid Cells Revealed by Deep Single-Cell RNA Sequencing[END_REF][START_REF] Matcovitch-Natan | Microglia development follows a stepwise program to regulate brain homeostasis[END_REF]. Thus, the percentage of microglia able to respond to a given neuromodulator may vary drastically depending on place, age and condition. A first requirement for being modulated by a neuromodulator in vivo is to be located close to its site of release. Pioneer electron microscopy studies provided ultrastructural evidence of contacts between non synaptic varicosities containing norepinephrine or 5-HT and glial processes [1,[START_REF] Ridet | Evidence for nonsynaptic serotonergic and noradrenergic innervation of the rat dorsal horn and possible involvement of neuron-glia interactions[END_REF][START_REF] Aoki | Beta-adrenergic receptors: astrocytic localization in the adult visual cortex and their relation to catecholamine axon terminals as revealed by electron microscopic immunocytochemistry[END_REF].

Recently, we quantified by confocal imaging and 3D reconstruction that, in the thalamus at P6, one microglia is surrounded by 43±7 serotonergic varicosities closer than 1 micrometer [15], a distance compatible with volume transmission. We observed similar features in different regions and at several developmental stages. Figure 1 illustrates an example of the proximity of microglia and serotonergic axons in the hippocampus of adult mice (Fig. 1A), with even some extremely close contacts (Fig. 1B). Close proximity of microglial processes and noradrenergic axons have been observed in the somatosensory cortex [START_REF] Liu | Neuronal network activity controls microglial process surveillance in awake mice via norepinephrine signaling[END_REF]. To our knowledge, similar studies have not yet been performed for the other neuromodulators. However, considering the density of dopaminergic, cholinergic and histaminergic fibers in sub-regions of the CNS and the mode of transmission of these neuromodulators, it is likely that microglial cells can detect them if they express the appropriate receptors. Thus, the second requirement for sensing neuromodulators is the expression of adequate receptors. In the next paragraphs and in Table 1, we will summarize what is currently known about this expression in microglia in vivo and in primary cultures.

Serotonin (5-HT)

In the brain, 5-HT is synthetized by serotonergic neurons of the raphe nuclei located in the brain stem, and released in close and distant regions of the whole CNS. 5-HT can also be released locally by mast cells, which are immune cells lining the outside of brain blood vessels [START_REF] Silver | Mast cells on the mind: new insights and opportunities[END_REF]. Fifteen subtypes of 5-HT receptors exist, divided in seven classes. While the 5-HT3 receptors are ion channels, all the others are G protein-coupled receptors (GPCRs). It has been shown by real time (RT-) and quantitative PCR that 5-HT2B receptors are the main serotonergic receptors expressed in primary microglial cultures [15,18]. Consistently, an agonist of 5-HT2B receptor elicits Ca 2+ responses in cultured microglia [18]. Expression of this receptor in microglia has also been confirmed at the neonatal [15] and adult stages [18] in vivo. In adulthood, 5-HT2B is consistently expressed in microglia throughout the brain, but additional serotonergic receptor subtypes may be expressed in specific regions [18].

Dopamine

Most of the dopaminergic neurons are clustered in the substantia nigra and the ventral tegmental area. Their axons project to many brain regions including the basal ganglia, the prefrontal cortex, the nucleus accumbens and the amygdala. Moreover, some dopaminergic neurons are located in the arcuate nucleus of the hypothalamus and project to the pituitary gland. Dopamine receptors are GPCRs classified into two major families, activating either Gs/olf (D1-like, i.e. D1 and D5 receptors) or Gi/o proteins (D2-like, i.e. D2, D3 and D4 receptors). The first direct evidence of the presence of dopamine receptors (D1, D2, D4, D5R) in rodent microglia in primary cultures and acute brain slices dates back to 2005 [19]. Human elderly microglial cultures have a similar but not identical (D1 to D4, not D5R) expression pattern [20]. Functionally, rat primary microglia cultures respond to a selective D2 receptor agonist, but not to D1R nor D3R agonists, by increasing tumor necrosis factor (TNF)-α mRNA [START_REF] Lewitus | Microglial TNF-α Suppresses Cocaine-Induced Plasticity and Behavioral Sensitization[END_REF]. The expression pattern of dopamine receptors might be region dependent Indeed, in rats, D1 and D2 receptors were observed in CD11b+ microglia isolated from the nucleus accumbens but not from the hippocampus [START_REF] Schwarz | FACS analysis of neuronal-glial interactions in the nucleus accumbens following morphine administration[END_REF]. It is also likely to be context dependent: according to immunohistochemistry and to a detailed examination of transgenic mice which express enhanced green fluorescent protein (EGFP) under the control of the Drd2a (D2R-encoding gene) promoter, D2 receptor is found in microglia activated by cerebral ischemia [23] but not in the healthy mouse cortex [24].

Norepinephrine

Norepinephrine is released by noradrenergic neurons, whose soma are located in the locus coeruleus in the brain stem. Their complex projections allow norepinephrine to be released in the whole CNS. Two classes of receptors for norepinephrine exist: the alpha (α1-A, B, C and α2-A, B, C) and beta (ß1, ß2 and ß3) adrenergic receptors, all GPCRs. In microglial primary culture, there is a consensus on the expression of the ß2 receptor subtype, based on mRNA expression analysis [START_REF] Tanaka | Existence of functional beta1-and beta2-adrenergic receptors on microglia[END_REF][START_REF] Mori | Effects of norepinephrine on rat cultured microglial cells that express alpha1, alpha2, beta1 and beta2 adrenergic receptors[END_REF][START_REF] Kong | Norepinephrine Promotes Microglia to Uptake and Degrade Amyloid ~ Peptide through Upregulation of Mouse Formyl Peptide Receptor 2 and Induction of Insulin-Degrading Enzyme[END_REF][START_REF] Gyoneva | Norepinephrine modulates the motility of resting and activated microglia via different adrenergic receptors[END_REF][START_REF] Ishii | Anti-inflammatory effects of noradrenaline on LPS-treated microglial cells: Suppression of NFκB nuclear translocation and subsequent STAT1 phosphorylation[END_REF] and/or pharmacological approaches [START_REF] Russo | Inhibition of microglial inflammatory responses by norepinephrine: effects on nitric oxide and interleukin-1beta production[END_REF][START_REF] Qian | β2-adrenergic receptor activation prevents rodent dopaminergic neurotoxicity by inhibiting microglia via a novel signaling pathway[END_REF][START_REF] Jiang | A novel role of microglial NADPH oxidase in mediating extra-synaptic function of norepinephrine in regulating brain immune homeostasis[END_REF][START_REF] Schneble | Phosphoinositide 3-kinase Î ties chemoattractant-and adrenergic control of microglial motility[END_REF]. Expression of ß2 adrenergic receptor in microglia in vivo was confirmed by transcriptome analysis on microglia acutely purified from the postnatal whole brain or from cortex and hippocampus [START_REF] Matcovitch-Natan | Microglia development follows a stepwise program to regulate brain homeostasis[END_REF]34]. The expression of ß1 is more controversial but has been reported in primary cultures [START_REF] Tanaka | Existence of functional beta1-and beta2-adrenergic receptors on microglia[END_REF][START_REF] Mori | Effects of norepinephrine on rat cultured microglial cells that express alpha1, alpha2, beta1 and beta2 adrenergic receptors[END_REF] and in vivo [START_REF] Matcovitch-Natan | Microglia development follows a stepwise program to regulate brain homeostasis[END_REF][START_REF] Hertz | Adrenoceptors in brain: cellular gene expression and effects on astrocytic metabolism and [Ca(2+)]i[END_REF]. Interestingly, the expression pattern of adrenergic receptors may depend on the physio-pathological state of the brain. Indeed, in microglial primary culture, treatment with LPS decreases the expression of ß2 and upregulates α2A adrenergic receptor [START_REF] Gyoneva | Norepinephrine modulates the motility of resting and activated microglia via different adrenergic receptors[END_REF][START_REF] Ishii | Anti-inflammatory effects of noradrenaline on LPS-treated microglial cells: Suppression of NFκB nuclear translocation and subsequent STAT1 phosphorylation[END_REF], which has a higher affinity for norepinephrine.

Acetylcholine (ACh)

Besides being a fast-acting neurotransmitter at the neuromuscular junction, ACh acts as neuromodulator in the CNS [START_REF] Picciotto | Acetylcholine as a neuromodulator: cholinergic signaling shapes nervous system function and behavior[END_REF]. The main source of ACh in the CNS are projecting neurons of discrete nuclei (pedunculopontine tegmental and lateral dorsal tegmental nuclei, medial habenula, etc.), which diffusely innervate the CNS. ACh is also released locally by cholinergic interneurons in the striatum and the nucleus accumbens. ACh receptors are classified into two main classes: nicotinic receptors (nAChRs), which are ionotropic cation channels permeable to Na + and K + , and muscarinic GPCRs (mAChRs). There are five subtypes of muscarinic receptors: M1, M3 and M5 coupled to Gq and M2 and M4 coupled to Gi/o proteins. The a7 nAChRs are the major components of the cholinergic anti-inflammatory pathway, a mechanism that down-regulates peripheral inflammation through ACh released by the vagus nerve that acts on macrophages through their a7 nAChRs [START_REF] Wang | Wang H_Nature_Nicotinic acetylcholine receptor a7 subunit is an essential regulator of inflammation[END_REF][START_REF] Rosas-Ballina | Cholinergic control of inflammation[END_REF]. Interestingly, a7 nAChRs are also present on microglia, in primary cultures [39][START_REF] Simone | Activation of α7 nicotinic acetylcholine receptor by nicotine selectively up-regulates cyclooxygenase-2 and prostaglandin E2 in rat microglial cultures[END_REF][START_REF] Suzuki | Microglial α7 nicotinic acetylcholine receptors drive a phospholipase C/IP3 pathway and modulate the cell activation toward a neuroprotective role[END_REF][START_REF] Morioka | Primary cultures of rat cortical microglia treated with nicotine increases in the expression of excitatory amino acid transporter 1 (GLAST) via the activation of the α7 nicotinic acetylcholine receptor[END_REF]. In vivo, the expression of nAChRs in microglia seems to increase upon inflammatory conditions. For instance, in an animal model of Alzheimer's disease, microglial a7 nAChR level, assessed by immunohistochemistry, strongly increases when activated microglia appear at sites of Ab deposition [START_REF] Matsumura | Temporal changes of CD68 and α7 nicotinic acetylcholine receptor expression in microglia in Alzheimer's disease-like mouse models[END_REF]. In human, in the brain of Alzheimer patients, a7 nAChRs are detected by immunohistochemistry in microglia surrounding the amyloid plaques [44]. Moreover, immunohistochemistry demonstrated overexpression of the a4b2 nAChRs in microglia after cerebral ischemia, consistent with in vivo increased positron emission tomography signal for a specific ligand [45]. The presence and function of metabotropic ACh receptors on microglia is poorly documented. Using mAChRs agonists in cultured microglia, two groups reported a transient increase in intracellular Ca 2+ [46,[START_REF] Whittemore | Carbachol increases intracellular free calcium in cultured rat microglia[END_REF]. Additionally, a flow cytometry analysis of mouse adult brain identified a M3-positive subpopulation representing around 15% of the microglial cells [8].

Histamine

In the brain, histamine is released by neurons from the tuberomammillary nucleus of the hypothalamus, or can be degranulated, like 5-HT, by activated mast cells [START_REF] Haas | Histamine in the nervous system[END_REF] (a role for this source of histamine will be illustrated in section 4). Moreover, microglia in primary cultures express histidine decarboxylase (Hdc), the enzyme responsible for histamine production, and Hdc level increases upon LPS treatment [START_REF] Iida | Histamine H 3receptor in primary mouse microglia inhibits chemotaxis, phagocytosis, and cytokine secretion[END_REF]. The previously mentioned investigation of microglial response to neuromodulators indicates that only 5 to 10% of cultured or freshly purified microglia respond to histamine by a Ca 2+ increase. This percentage reaches 60% in cells treated for 24h with LPS [9]. Four receptors exist for histamine, H1 to H4, all GPCRs. Bulk transcriptome analyses on acutely purified microglia report the expression mainly of Hrh2 [START_REF] Matcovitch-Natan | Microglia development follows a stepwise program to regulate brain homeostasis[END_REF]34], especially in cortical microglia [START_REF] Grabert | Microglial brain region-dependent diversity and selective regional sensitivities to aging[END_REF]. In primary cultures, mRNA expression analysis as well as pharmacological and functional approaches support the expression of Hrh3 [START_REF] Iida | Histamine H 3receptor in primary mouse microglia inhibits chemotaxis, phagocytosis, and cytokine secretion[END_REF], Hrh1 and Hrh4 genes [START_REF] Lenz | Mast Cells in the Developing Brain Determine Adult Sexual Behavior[END_REF].

Acute modulation of microglia by neuromodulators: effects on morphology, motility and phagocytic activity

Dynamic interactions between microglia and other cells rely on the motility of their fine processes, as well as their migration and phagocytic activity. We will hereby summarize how these functions can be rapidly regulated by neuromodulators (Fig. 2).

Serotonin (5-HT)

We demonstrated that a local application of 5-HT on acute brain slices induces a directional motility of microglial processes, mediated by the microglial 5-HT2B receptor [15,[START_REF] Etienne | Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices[END_REF]. This is consistent with a previous study reporting that 5-HT promotes the increase of microglia processes motility in response to a laser lesion in acute brain slices, and enhances the chemotactic effect of ATP in microglia primary cultures [18]. The same study described an inhibitory effect of high concentrations of 5-HT or 5-HT2 agonists on the phagocytosis of artificial microspheres by microglia in primary cultures and in acute brain slices [18].

Dopamine

In primary microglial cultures, dopamine reduces the number of cellular processes and increases microglial cell area in basal conditions [START_REF] Fan | Differential Regulation of Adhesion and Phagocytosis of Resting and Activated Microglia by Dopamine[END_REF]. Regarding microglial migration, it is enhanced by dopamine and D1-and D2-receptor agonists in rodent cultures [19] as well as in human elderly microglial cultures [20].

Norepinephrine

The effect of norepinephrine on microglial morphology has been first studied on acute brain slices and on primary microglia cultivated in a tridimensional matrix. In these experimental models, norepinephrine, through activation of ß2 adrenergic receptors, induces a reduction of microglial arborization within minutes, and inhibits the effect of ATP on microglial processes outgrowth [START_REF] Gyoneva | Norepinephrine modulates the motility of resting and activated microglia via different adrenergic receptors[END_REF]. Interestingly, retraction of microglial processes upon norepinephrine application is also seen in microglia stimulated by LPS for 24h, but this effect is mediated by α2A adrenergic receptors. As ß2 and α2A are likely to have different coupling (Gs and Gi, respectively, in non-microglial cells), and norepinephrine does not induce Ca 2+ rise [START_REF] Gyoneva | Norepinephrine modulates the motility of resting and activated microglia via different adrenergic receptors[END_REF], the signaling pathways mediating the retraction of microglial processes remain to be elucidated. Recently, the control of microglia morphology by norepinephrine has been confirmed in vivo by two independent studies. Both showed that in awake mice, norepinephrine tonically limits, though activation of ß2 receptors, the arborization and the motility of microglia, thus suppressing their surveillance of the parenchyma. This suppressive effect is abolished, and thus surveillance increases, in the presence of anesthetic agents which inhibit noradrenergic neurons activity in the locus coeruleus [START_REF] Liu | Neuronal network activity controls microglial process surveillance in awake mice via norepinephrine signaling[END_REF][START_REF] Stowell | Noradrenergic signaling in the wakeful state inhibits microglial surveillance and synaptic plasticity in the mouse visual cortex[END_REF] . Further experiments will be required to determine whether a physiological reduction of noradrenergic neurons activity, e.g. during sleep, also significantly increases microglial surveillance. To note is that under anesthesia, when microglia are more ramified and motile than during wakefulness, activation of ß2 receptors prevents the recruitment of microglial processes toward a focal lesion injury [START_REF] Stowell | Noradrenergic signaling in the wakeful state inhibits microglial surveillance and synaptic plasticity in the mouse visual cortex[END_REF]. This effect is reminiscent of the inhibitory effect of ß2 receptors agonists on microglial processes attraction toward ATP, observed in acute slices and mentioned above [START_REF] Gyoneva | Norepinephrine modulates the motility of resting and activated microglia via different adrenergic receptors[END_REF]. Overall, these results suggest that, in vivo, variations in norepinephrine tone upon stress or sleep-wake cycle may alter microglial interaction with synapses, neurons, vasculature and responsiveness to damaged tissues. In primary cultures, norepinephrine does not seem to promote nor inhibit migration of microglial cells when applied alone [19], but modulatory effects on chemotaxis induced by other compounds have been reported. On one hand, high doses (30 or 100 µM) of norepinephrine inhibit chemotaxis toward ATP [START_REF] Gyoneva | Norepinephrine modulates the motility of resting and activated microglia via different adrenergic receptors[END_REF] and C5a complement [START_REF] Schneble | Phosphoinositide 3-kinase Î ties chemoattractant-and adrenergic control of microglial motility[END_REF]. The latter effect is likely to occur through ß2 adrenergic receptors and activation of adenylate cyclase, protein kinase A (PKA) and phosphoinositide 3 kinase (PI3K). On the other hand, low doses of norepinephrine or ß2 adrenergic agonist potentiate chemotaxis toward TNF-α or amyloid (A)ß peptide [START_REF] Heneka | Locus ceruleus controls Alzheimer's disease pathology by modulating microglial functions through norepinephrine[END_REF]. This discrepancy might come from the different attractants used, the dose of adrenergic agonists, or both. In vivo, in a mouse model of Alzheimer's disease, transplanted microglia fail to migrate toward amyloid plaques if the terminals from the locus coeruleus neurons have been depleted of norepinephrine [START_REF] Heneka | Locus ceruleus controls Alzheimer's disease pathology by modulating microglial functions through norepinephrine[END_REF]. This supports a positive role of norepinephrine on microglial migration in vivo, at least in this pathological context. In primary microglial cultures, norepinephrine potentiates the phagocytosis and degradation of Aß peptide, through ß2 adrenergic receptors [START_REF] Kong | Norepinephrine Promotes Microglia to Uptake and Degrade Amyloid ~ Peptide through Upregulation of Mouse Formyl Peptide Receptor 2 and Induction of Insulin-Degrading Enzyme[END_REF][START_REF] Heneka | Locus ceruleus controls Alzheimer's disease pathology by modulating microglial functions through norepinephrine[END_REF]. This is consistent with in vivo observations in models of Alzheimer's disease. Indeed, toxin-based depletion of norepinephrine reduces the number of microglia around amyloid plaques and their content in Aß peptide, and injury of the locus coeruleus increases amyloid plaques deposit and glial inflammation [START_REF] Heneka | Locus ceruleus controls Alzheimer's disease pathology by modulating microglial functions through norepinephrine[END_REF][START_REF] Heneka | Locus ceruleus degeneration promotes Alzheimer pathogenesis in amyloid precursor protein 23 transgenic mice[END_REF]. Altogether, this suggests a defect in Aß elimination in vivo when norepinephrine level is reduced.

Acetylcholine (ACh)

To our knowledge, the potential effect of ACh on microglial migration has not been investigated per se; however, it has been shown that agonists of mACh receptors such as carbachol induce chemotaxis in primary cultures of microglia [8]. Regarding phagocytosis, metabotropic and ionotropic receptors of ACh may have different effects, or their effect might depend on the target to be phagocytosed. Indeed, on one hand, carbacholan agonist of mAChR-diminishes the phagocytosis of latex beads in primary cultures of microglia [8]. On the other hand, galantamine -an allosteric activator of a7 nAChR-significantly enhances the phagocytosis of Aβ by rat primary cultures of microglia in presence of an orthosteric ligand of a7 nAChRs [44]. Thus, mAChRs and nAChRs seem to have opposite effects on phagocytosis in primary cultures, and it would be interesting to see if the expression of these receptors is coordinately regulated in vivo to promote one effect or the other.

Several efforts were made to characterize the ACh-and nicotine-induced regulation of intracellular signaling pathways. Both ACh and nicotine lead to a transient increase in intracellular Ca 2+ [9,[START_REF] Suzuki | Microglial α7 nicotinic acetylcholine receptors drive a phospholipase C/IP3 pathway and modulate the cell activation toward a neuroprotective role[END_REF]. However, surprisingly -considering the ionotropic nature of nAChR-, nicotine-induced intracellular Ca 2+ increase is independent of extracellular Ca 2+ , and blocked by inhibitors of phospholipase C or inositol phosphate 3 receptor. This supports the hypothesis that a7 nAChRs in microglia may not function only as conventional ion channels, but rather drive a signaling cascade triggering Ca 2+ release from intracellular stores [START_REF] Suzuki | Microglial α7 nicotinic acetylcholine receptors drive a phospholipase C/IP3 pathway and modulate the cell activation toward a neuroprotective role[END_REF].

Histamine

Histamine triggers motility of microglia out of cortical explants, in a H4R-dependent manner [START_REF] Ferreira | Histamine modulates microglia function[END_REF], but it inhibits, through H3R, ATP-induced chemotaxis in primary cultures [START_REF] Iida | Histamine H 3receptor in primary mouse microglia inhibits chemotaxis, phagocytosis, and cytokine secretion[END_REF]. These apparently opposite effects could be imputable to the different experimental models (tissue explants vs primary cultures) or the different read-outs used (spontaneous migration vs chemotaxis). Regarding phagocytosis, incubation for one hour with histamine or a H3R agonist inhibits phagocytosis of artificial microspheres by primary cultures of microglia [START_REF] Iida | Histamine H 3receptor in primary mouse microglia inhibits chemotaxis, phagocytosis, and cytokine secretion[END_REF].

3.

Involvement of neuromodulators in the regulation of microglia immune polarization Microglia show a continuum of activation states (reviewed in [START_REF] Ransohoff | A polarizing question: Do M1 and M2 microglia exist[END_REF]). Depending on age and context, microglia can indeed release a vast array of factors with pro-or anti-inflammatory activity, neurotoxic or neuroprotective effects. They can also express a considerable variety or receptors, and differ by their phagocytic capacity. Transition from one state to another can be triggered by bacterial or viral compounds, or by endogenous factors like cytokines, aggregated proteins, etc. In this section and in Figure 2, we will show how neuromodulators also endorse a role of "immune" modulators by regulating the polarization of microglia toward different activation states.

Serotonin (5-HT)

A role of 5-HT in microglia functional polarization is supported by the observation that lack of 5-HT2B receptor induces the overexpression of some cytokines and cytokine receptors by microglia in primary cultures [15]. In addition, in a model of amyotrophic lateral sclerosis, microglia adopt an abnormal phenotype if mice are also knocked-out for this receptor gene [START_REF] Oussini | Serotonin 2B receptor slows disease progression and prevents degeneration of spinal cord mononuclear phagocytes in amyotrophic lateral sclerosis[END_REF], but the implication of the microglial 5-HT2B receptor remains to be demonstrated. This would be consistent with a tonic role of 5-HT in preventing microglia activation, as shown for human peripheral macrophages [START_REF] De Las Casas-Engel | Serotonin Skews Human Macrophage Polarization through HTR2B and HTR7[END_REF]. 5-HT might also regulate the dialog between microglia and other cells by stimulating the release of exosomes from microglial primary cultures [START_REF] Glebov | Serotonin stimulates secretion of exosomes from microglia cells[END_REF].

Dopamine

In primary microglial cultures, dopamine generally exerts a protective effect against LPS-induced inflammation, attenuating the release of a prominent indicator of microglial activation, nitric oxide [19]. This anti-inflammatory action of dopamine might be imputable to inhibition of the Angiotensin-1/NADPH-oxidase/superoxide axis [START_REF] Dominguez-Meijide | Dopamine modulates astroglial and microglial activity via glial renin-angiotensin system in cultures[END_REF]. In vivo, an increase of dopamine extracellular level in the striatum, in response to cocaine, stimulates microglia. Indeed, these elevated dopamine levels not only activate D1 receptors on striatal neurons, but also target D2 receptors expressed by microglia. This increases TNF-α mRNA level in these cells [START_REF] Lewitus | Microglial TNF-α Suppresses Cocaine-Induced Plasticity and Behavioral Sensitization[END_REF]. TNF-α, besides its widely known pro-inflammatory effects, is a potent inducer of synaptic changes [START_REF] Lewitus | An adaptive role of TNFα in the regulation of striatal synapses[END_REF][START_REF] Beattie | Control of synaptic strength by glial TNFα[END_REF], which here alleviate drug-induced behavioral alterations such as cocaine sensitization [START_REF] Lewitus | Microglial TNF-α Suppresses Cocaine-Induced Plasticity and Behavioral Sensitization[END_REF]. These results suggest that modulation of microglia might be of therapeutical interest in drug addiction.

Norepinephrine

In primary cultures of microglia, norepinephrine counteracts the effect of LPS or Aß peptide on the expression or release of TNF-α, IL-1ß, IL-6, IL-12p40, nitric oxide and reactive oxygen species [19,[START_REF] Mori | Effects of norepinephrine on rat cultured microglial cells that express alpha1, alpha2, beta1 and beta2 adrenergic receptors[END_REF][START_REF] Ishii | Anti-inflammatory effects of noradrenaline on LPS-treated microglial cells: Suppression of NFκB nuclear translocation and subsequent STAT1 phosphorylation[END_REF][START_REF] Qian | β2-adrenergic receptor activation prevents rodent dopaminergic neurotoxicity by inhibiting microglia via a novel signaling pathway[END_REF][START_REF] Jiang | A novel role of microglial NADPH oxidase in mediating extra-synaptic function of norepinephrine in regulating brain immune homeostasis[END_REF][START_REF] Heneka | Locus ceruleus controls Alzheimer's disease pathology by modulating microglial functions through norepinephrine[END_REF][START_REF] Madrigal | Norepinephrine protects cortical neurons against microglial-induced cell death[END_REF][START_REF] Yao | Lesion of the locus coeruleus aggravates dopaminergic neuron degeneration by modulating microglial function in mouse models of Parkinson׳s disease[END_REF]. In addition, it limits LPS-induced toxicity of microglia toward cortical [START_REF] Madrigal | Norepinephrine protects cortical neurons against microglial-induced cell death[END_REF] and dopaminergic neurons [START_REF] Qian | β2-adrenergic receptor activation prevents rodent dopaminergic neurotoxicity by inhibiting microglia via a novel signaling pathway[END_REF][START_REF] Jiang | A novel role of microglial NADPH oxidase in mediating extra-synaptic function of norepinephrine in regulating brain immune homeostasis[END_REF]. The signaling pathway linking norepinephrine to inhibition of microglial activation in culture is unclear, as well as the signal transduction for its effects on microglial processes (section 2). Pharmacological approaches generally point to a role of ß2 adrenergic receptors, whose canonical coupling is Gs. However, several studies deny a role of cyclic adenosine monophosphate (cAMP)and PKA-dependent, as well as Ca 2+ -mediated, pathways, in these effects of norepinephrine [START_REF] Gyoneva | Norepinephrine modulates the motility of resting and activated microglia via different adrenergic receptors[END_REF][START_REF] Ishii | Anti-inflammatory effects of noradrenaline on LPS-treated microglial cells: Suppression of NFκB nuclear translocation and subsequent STAT1 phosphorylation[END_REF][START_REF] Qian | β2-adrenergic receptor activation prevents rodent dopaminergic neurotoxicity by inhibiting microglia via a novel signaling pathway[END_REF][START_REF] Jiang | A novel role of microglial NADPH oxidase in mediating extra-synaptic function of norepinephrine in regulating brain immune homeostasis[END_REF][START_REF] Xu | Enriched environment enhances βadrenergic signaling to prevent microglia inflammation by~amyloid-β[END_REF]. Non-G protein coupling would be in accordance with the observation that ß2 and α2A adrenergic receptors have the same morphological effect on microglia [START_REF] Gyoneva | Norepinephrine modulates the motility of resting and activated microglia via different adrenergic receptors[END_REF], although they are supposed to have opposite effects on adenylate cyclase. An alternative pathway could be mediated by β arrestin-2 [START_REF] Qian | β2-adrenergic receptor activation prevents rodent dopaminergic neurotoxicity by inhibiting microglia via a novel signaling pathway[END_REF]. To note is that a report, furthermore, questions the involvement of adrenergic receptors themselves in some effects of norepinephrine on microglia [START_REF] Jiang | A novel role of microglial NADPH oxidase in mediating extra-synaptic function of norepinephrine in regulating brain immune homeostasis[END_REF]. In vivo, decreasing norepinephrine levels potentiates brain inflammation in rodent models of Parkinson's [START_REF] Yao | Lesion of the locus coeruleus aggravates dopaminergic neuron degeneration by modulating microglial function in mouse models of Parkinson׳s disease[END_REF] and Alzheimer's diseases [START_REF] Heneka | Locus ceruleus degeneration promotes Alzheimer pathogenesis in amyloid precursor protein 23 transgenic mice[END_REF], and in response to systemic inflammation [START_REF] Bharani | A noradrenergic lesion aggravates the effects of systemic inflammation on the hippocampus of aged rats[END_REF]. Although non-microglial noradrenergic-sensitive cells, like astrocytes, could participate to this neuroprotective and anti-inflammatory effect of norepinephrine [START_REF] Madrigal | Norepinephrine protects cortical neurons against microglial-induced cell death[END_REF], these experiments are consistent with the results obtained on primary cultures of microglia. This anti-inflammatory role of norepinephrine may also explain in part why, in Parkinson's and Alzheimer's patients, neuronal loss in the locus coeruleus -where noradrenergic neurons are located-occurs prior to neurodegeneration in other brain regions [START_REF] Braak | Staging of brain pathology related to sporadic Parkinson's disease[END_REF][START_REF] Zarow | Neuronal loss is greater in the locus coeruleus than nucleus basalis and substantia nigra in Alzheimer and Parkinson diseases[END_REF].

Acetylcholine (ACh)

In the periphery, macrophages are controlled by ACh released by the vagus nerve and acting onto their a7 nAChRs (a mechanism called "cholinergic anti-inflammatory pathway") [START_REF] Wang | Wang H_Nature_Nicotinic acetylcholine receptor a7 subunit is an essential regulator of inflammation[END_REF][START_REF] Rosas-Ballina | Cholinergic control of inflammation[END_REF]. Similarly, the role of nAChRs in modulating microglia activation state has been broadly investigated. A short pretreatment of rodent microglial primary cultures with ACh or nicotine drastically reduces their response to LPS in terms of mitogen-activated protein kinase (MAPK) activation and expression or release of pro-inflammatory factors, such as TNF-α [39][START_REF] Simone | Activation of α7 nicotinic acetylcholine receptor by nicotine selectively up-regulates cyclooxygenase-2 and prostaglandin E2 in rat microglial cultures[END_REF][START_REF] Suzuki | Microglial α7 nicotinic acetylcholine receptors drive a phospholipase C/IP3 pathway and modulate the cell activation toward a neuroprotective role[END_REF]. Such pretreatment also increases the production of anti-inflammatory factors such as prostaglandin (PG) E2 [START_REF] Simone | Activation of α7 nicotinic acetylcholine receptor by nicotine selectively up-regulates cyclooxygenase-2 and prostaglandin E2 in rat microglial cultures[END_REF]. Transcriptomic analyses on primary microglial cultures from sheep fetal brain are in line with these results. In this animal model, α7nAChR agonists limit the acquisition of a pro-inflammatory phenotype in response to LPS, while antagonizing α7nAChRs does the opposite [START_REF] Cortes | α7 nicotinic acetylcholine receptor signaling modulates the inflammatory phenotype of fetal brain microglia: First evidence of interference by iron homeostasis[END_REF]. Eventually, stimulation of microglial a7 nAChR was shown to limit the neurotoxicity of LPS-activated microglia on neurons cultured from hippocampus and cortex [START_REF] Noda | Nicotine inhibits activation of microglial proton currents via interactions with α7 acetylcholine receptors[END_REF]. A neuroprotective effect of a7 nAChR stimulation, microgliadependent, was also uncovered in models of stroke in organotypic slices and in vivo [START_REF] Parada | The Microglial α7-Acetylcholine Nicotinic Receptor Is a Key Element in Promoting Neuroprotection by Inducing Heme Oxygenase-1 via Nuclear Factor Erythroid-2-Related Factor 2[END_REF].

Beside its immunomodulatory action, stimulation of a7 nAChR might be neuroprotective by increasing the expression of the glutamate transporter GLAST in microglia, thereby maintaining glutamate extracellular concentration below its excitotoxic threshold [START_REF] Morioka | Primary cultures of rat cortical microglia treated with nicotine increases in the expression of excitatory amino acid transporter 1 (GLAST) via the activation of the α7 nicotinic acetylcholine receptor[END_REF].

Histamine

In primary microglia, histamine can promote the release of PGE2 by microglia [START_REF] Lenz | Mast Cells in the Developing Brain Determine Adult Sexual Behavior[END_REF], which is synaptogenic on neurons of the preoptic area but not in the hippocampus [START_REF] Amateau | A novel mechanism of dendritic spine plasticity involving estradiol induction of prostaglandin-E2[END_REF]. This will be further discussed in section 4. In vivo, several studies report a protective and anti-inflammatory effect of antagonists of histamine receptors in various pathologies such as Alzheimer's and Parkinson's disease and multiple sclerosis (reviewed in [START_REF] Silver | Mast cells on the mind: new insights and opportunities[END_REF][START_REF] Rocha | Histamine: a new immunomodulatory player in the neuron-glia crosstalk[END_REF]). Noteworthy, histamine receptors are expressed in various cell types of the brain and of the peripheral immune system. Studies on mutants conditionally invalidated for histamine receptors in microglia only will therefore be needed to definitely implicate these cells in the effects of histamine receptor ligands.

4.

Microglia as effectors of neuromodulators Many events can affect the extracellular level of neuromodulators in the CNS and thus trigger changes in microglia. During development, this level will regionally vary according to the ontogeny of each of the neuromodulatory systems. It will particularly depend on the growth of the axons releasing the neuromodulators, and on the maturation of their release and reuptake machineries. For 5-HT, transient and ectopic expression of 5-HT transporter (SERT) in non-serotonergic neurons may impact on local availability of 5-HT [START_REF] Gaspar | The developmental role of serotonin: news from mouse molecular genetics[END_REF]. On a different time-scale, the levels of histamine, 5-HT and norepinephrine undergo circadian variations, with lower levels during sleep than wake phases. External cues or pathologies superimpose their effects onto these physiological variations. To provide a few examples, peripheral inflammation increases the level of 5-HT and dopamine in the brain for a few hours [START_REF] Guo | Quantitative profiling of neurotransmitter abnormalities in the hippocampus of rats treated with lipopolysaccharide: Focusing on kynurenine pathway and implications for depression[END_REF]; norepinephrine is released upon stress; antidepressant treatments with selective 5-HT or norepinephrine reuptake inhibitors, or with monoamine oxidase inhibitors, increase the level of 5-HT, norepinephrine or other monoamines, by decreasing their reuptake or degradation, respectively. Based on the evidences presented in the previous sections, these variations can induce transient or long-lasting changes in microglia. Whether these microglial changes are required to mediate some of the neuronal and behavioral effects induced by these variations remains to be fully understood. We hereby discuss few recent works which support the hypothesis that regulation of microglia by neuromodulators is needed for proper CNS development and the control of higher brain functions in physiological or pathological conditions.

Histamine, microglia, and masculinization of the brain

Sexual dimorphism exists in the brain as in other organs and results from developmental programs regulated by sexual hormones. In adult rodents, one difference between male and female brains is a larger number of dendritic spines in the preoptic area (POA) of males, a hypothalamic region essential for expression of copulatory behavior. It results from an increased synaptogenesis during neonatal development, which is triggered by PGE2 locally produced under the control of estradiol [START_REF] Amateau | A novel mechanism of dendritic spine plasticity involving estradiol induction of prostaglandin-E2[END_REF][START_REF] Amateau | Induction of PGE2 by estradiol mediates developmental masculinization of sex behavior[END_REF]. It is known that when sexual dimorphism appears in the POA of neonates, microglial cells are more ameboid, and probably more "active" in males than in females [START_REF] Lenz | Microglia are essential to masculinization of brain and behavior[END_REF]. Recently, it was proposed that microglia are the source of PGE2, but under the intermediate control of histamine. Indeed, manipulating histamine release by mast cells during the neonatal period impacts on the levels of PGE2 in the POA and on the establishment of male sexual behavior. Moreover, using combinations of primary cultures from different cell types (microglia, mast cells, neurons), Lenz and colleagues demonstrated that estradiol induces the degranulation of histamine by mast cells, which, in turn, activates H1R and H4R on microglia and potentiates their release of PGE2 [START_REF] Lenz | Mast Cells in the Developing Brain Determine Adult Sexual Behavior[END_REF]. PGE2, lastly, promotes dendritic spines formation. Thus, microglia could be effectors of histamine for the release of PGE2 and the masculinization of brain and behavior, but additional experiments are required to demonstrate that this regulation occurs identically in vivo.

Norepinephrine, microglia, and the neuroprotective effect of an enriched environment

Enriching the environment of rodents is a paradigm of cognitive reserve in humans. Indeed, an enriched environment promotes rodent's resistance to neuronal and cognitive deficits and limits microglial activation in models of Alzheimer's disease [START_REF] Xu | Environmental Enrichment Potently Prevents Microglia-Mediated Neuroinflammation by Human Amyloid β-Protein Oligomers[END_REF]. ß2 adrenergic receptor was known to be required for some of the neuroprotective effects of an enriched environment [START_REF] Li | Environmental novelty activates β2-adrenergic signaling to prevent the impairment of hippocampal LTP by Aβ oligomers[END_REF].A recent study unraveled its additional role in limiting the activation of microglia in a model of Alzheimer's disease induced by human Aß oligomers injection [START_REF] Xu | Enriched environment enhances βadrenergic signaling to prevent microglia inflammation by~amyloid-β[END_REF]. Indeed, in this model, the anti-inflammatory effect of an enriched environment is suppressed by treatment with ß-adrenergic antagonists. It is also absent in knock-outs of ß1/ß2 adrenergic receptors. Conversely, the inhibition of microglia activation can be mimicked by administration of ß-adrenergic agonists. In addition, the authors show that administration of Aß oligomers decreases the expression of ß-adrenergic receptors in microglia as well as the extracellular content in norepinephrine, and that both alterations are absent in mice raised in an enriched environment [START_REF] Xu | Enriched environment enhances βadrenergic signaling to prevent microglia inflammation by~amyloid-β[END_REF]. In summary, these data are consistent with a model where activation of microglial ß2 adrenergic receptors prevents Alzheimer's disease progression. In this model, the beneficial effect of an enriched environment would be to sustain the expression of microglial ß2 adrenergic receptors, which tends to decrease in the presence of Aß oligomers. Although an analysis of mice specifically invalidated for microglial ß2 adrenergic receptors would be required to validate this model, these data support the idea that regulation of microglia by norepinephrine is relevant for the maintenance of brain homeostasis in a pathological context.

Conclusion and perspectives

Here, we first summarized the evidence that microglial cells are equipped to respond to neuromodulators. We then illustrated how neuromodulators can impact rapidly on microglial processes motility and phagocytosis, and more durably on their immune polarization. These regulations of microglial cells can, in turn, impact on neurons by modulating synapse formation, neuronal survival, etc. Altogether, these experiments support the hypothesis that some of the effects of the neuromodulators on CNS development and homeostasis may be performed through the modulation of microglia. There are now indications that this could be the case for some developmental functions, like masculinization of the brain, and for the neuroprotective effect of enriched environment. Nonetheless, the involvement of microglia in many other potential roles of neuromodulators, for example on mood, sleep-wake cycle or response to inflammation, remain to be addressed. To note is that several of the regulations described in this review have been demonstrated on cultivated or freshly purified microglia and need to be confirmed in vivo, by inhibiting or activating receptors selectively in microglia. To do this, crossing transgenic mouse lines is to date the most straightforward but time-consuming approach. Hopefully, the development of better experimental systems, such as microglial targeting with dendrimers and induced pluripotent stem cells (IPSc)derived microglia that can be more easily genetically engineered, will allow to further explore this exciting research area in the near future. (1a) On the one hand, clearance of artificial beads is reduced after application of 5-HT [18], histamine [START_REF] Iida | Histamine H 3receptor in primary mouse microglia inhibits chemotaxis, phagocytosis, and cytokine secretion[END_REF], and carbachol, an agonist of mACh receptors [8]; (1b) on the other hand, phagocytosis and degradation of Aβ are increased by norepinephrine acting on β2 receptors [START_REF] Kong | Norepinephrine Promotes Microglia to Uptake and Degrade Amyloid ~ Peptide through Upregulation of Mouse Formyl Peptide Receptor 2 and Induction of Insulin-Degrading Enzyme[END_REF][START_REF] Heneka | Locus ceruleus controls Alzheimer's disease pathology by modulating microglial functions through norepinephrine[END_REF] and ACh acting on α7 nAChR [44]. 2: To scan the environment and make physical interactions, microglia have to be highly motile, extending and retracting their fine processes. (2a) In normal conditions, besides potentiating ATP-induced increase in microglia process motility (not illustrated here [18]), local applications of 5-HT on acute brain slices induces a directional extension of microglial processes, an effect dependent on 5-HT2B receptors [15]. Norepinephrine instead promotes retraction of microglial processes through β2 receptors [28][16,53] and prevents ATP-or lesion-induced motility (not illustrated here [28][53]). (2b) In microglia treated with LPS, norepinephrine induces a reduction of microglial arborization, this time mediated by α2A receptors [START_REF] Gyoneva | Norepinephrine modulates the motility of resting and activated microglia via different adrenergic receptors[END_REF]. 3 and 4: Several experiments demonstrate that neuromodulators actively regulate microglial immune polarization and influence their release of soluble factors. 3: Some of these factors are released by microglia in basal conditions, such as PGE2, whose release is promoted by histamine [START_REF] Lenz | Mast Cells in the Developing Brain Determine Adult Sexual Behavior[END_REF], or TNF-α, whose release is promoted by dopamine in the striatum [START_REF] Lewitus | An adaptive role of TNFα in the regulation of striatal synapses[END_REF]. 4: However, in a number of pathological conditions, microglia can become neurotoxic and release pro-inflammatory cytokines, factors and reactive oxygen species. Most neuromodulators, including dopamine, norepinephrine and ACh, exert a protective action by inhibiting this polarization of microglia toward a toxic phenotype after exposure to inflammatory stimuli (numerous references, cited in section 3). 5: Eventually, "danger" or inflammatory signals promote microglial migration and process outgrowth, which are potentiated by 5-HT [18] and agonists of mAChR [8], and inhibited by histamine [START_REF] Iida | Histamine H 3receptor in primary mouse microglia inhibits chemotaxis, phagocytosis, and cytokine secretion[END_REF]. Norepinephrine modulates microglial chemotaxis toward danger signals in a dual way: high doses inhibit process outgrowth toward ATP and C5 complement [START_REF] Gyoneva | Norepinephrine modulates the motility of resting and activated microglia via different adrenergic receptors[END_REF][START_REF] Schneble | Phosphoinositide 3-kinase Î ties chemoattractant-and adrenergic control of microglial motility[END_REF], but low doses increase chemotaxis toward Aβ and TNF-α [START_REF] Heneka | Locus ceruleus controls Alzheimer's disease pathology by modulating microglial functions through norepinephrine[END_REF]. 5-HT: serotonin; α2A: α2A adrenergic receptors; ß2: ß2 adrenergic receptors; Aβ: amyloid β; ACh: acetylcholine; ATP: adenosine triphosphate; C5: C5 complement protein; LPS: lipopolysaccharide; mAChR: muscarinic acetylcholine receptors; nAChR: nicotinic acetylcholine receptors; PGE2: prostaglandin E2; TNF-α: tumor necrosis factor α.

Figure 1 :

 1 Figure1: Close contacts between microglial processes and serotonergic varicosities in the adult brain. (A) Projection of a 20 µm-thick stack of confocal images from the hippocampus of an adult mouse, illustrating proximity of microglia (red, stained with anti-Iba1 antibody) and serotonergic axons (green, stained with anti-serotonin antibody; note the typical varicosities along the axons). Noteworthy, to estimate distances and visualize appositions, a projection image is not sufficient, and 3D reconstruction is required. Scale bar: 10 µm. (B) Imaris 3D reconstruction from a stack of confocal images allows to show some close appositions (square) of microglial processes and serotonergic axons, in addition to the general presence of varicosities at a micrometer-range distance of microglia. The discontinuous appearance of the serotonergic axons in the reconstruction is due to the contrast between the strongly stained varicosities, where serotonin-containing vesicles accumulate, and the rest of the axons. Nuclei, stained with Bis-Benzimide, are in blue. Left: overview, right: detail showing a microglia process surrounding a serotonin varicosity (asterisk). Scale bars: 5 µm.

Figure 2 .

 2 Figure 2. Regulation of microglial functions by neuromodulators.Microglia can act on neurons and other cell types through physical interactions and by releasing a vast array of soluble factors, and neuromodulators were shown to regulate many of these processes. 1: Phagocytosis of dying neurons, excessive synapses or unwanted compounds is pivotal during CNS development and to maintain CNS homeostasis. Several neuromodulators can regulate phagocytosis, although the final effect seems to be dependent on the target to be phagocytosed. (1a) On the one hand, clearance of artificial beads is reduced after application of 5-HT[18], histamine[START_REF] Iida | Histamine H 3receptor in primary mouse microglia inhibits chemotaxis, phagocytosis, and cytokine secretion[END_REF], and carbachol, an agonist of mACh receptors[8]; (1b) on the other hand, phagocytosis and degradation of Aβ are increased by norepinephrine acting on β2 receptors[START_REF] Kong | Norepinephrine Promotes Microglia to Uptake and Degrade Amyloid ~ Peptide through Upregulation of Mouse Formyl Peptide Receptor 2 and Induction of Insulin-Degrading Enzyme[END_REF][START_REF] Heneka | Locus ceruleus controls Alzheimer's disease pathology by modulating microglial functions through norepinephrine[END_REF] and ACh acting on α7 nAChR[44]. 2: To scan the environment and make physical interactions, microglia have to be highly motile, extending and retracting their fine processes. (2a) In normal conditions, besides potentiating ATP-induced increase in microglia process motility (not illustrated

Table 1 : Expression of receptors for neuromodulators in microglia. Biological material:

 1 C: primary Culture of microglia, FP: Freshly Purified microglia, S: brain Slices, IV: In Vivo. When not specified, experiments have been performed on rodents.

	Receptor	Evidence for the expression of the receptors in microglia	References,
	subtypes		biological
			material
	Histamine
	?	Histamine application induces a calcium increase in 5-10% of freshly	[9] [C, FP]
		purified or cultured microglia (60% if cells were pretreated with LPS
		for 24h)
	H1, H4	Antagonists for H1R and H4R limit the effects of mast cells	[50] [C]
		supernatant on the production of PGE2 by microglia
	H2	RNA-Seq	[11,14,34]
			[FP]
	H3	H3R agonist has similar effects than histamine on phagocytosis,	[49] [C]
		chemotaxis by ATP and response to LPS
	Norepinephrine
	ß	ß adrenergic receptors antagonists have the same effect than	[54] [C]
		norepinephrine on cytokine release, phagocytosis, migration, in
		response to amyloid peptide
	ß	An agonist of ß adrenergic receptors has the same inhibitory effect	[33] [C]
		than norepinephrine on migration induced by C5a
	a1 and 2, ß1 and	RT-PCR	[26] [C]
	2
	a, ß1 and 2	The anti-inflammatory effect of norepinephrine on LPS-treated	[19] [C]
		microglia is blocked by ß1 and ß2 adrenergic receptors antagonists;
		membrane currents are modulated by norepinephrine and by α-and
		β-adrenergic agonists
	ß 1 and 2	RT-PCR; norepinephrine and agonists of ß1 or ß2 induce cAMP, and	[25] [C]
		antagonists of ß1 or ß2 block the induction of cAMP by
		norepinephrine
	ß 1 and 2	RNA-Seq (found in microglia purified from adult, but not newborn,	[14] [FP]
		animals)
	ß 1 and 2	A ß-adrenergic agonist prevents the induction of cytokines by amyloid	[66] [C, FP]
		peptide; Adrb1 and Adrb2 genes are detected by QPCR and ß2 is
		detected by Western-Blot
	ß2	QPCR	[27] [C]

This definition of neuromodulators excludes adenosine triphosphate (ATP) and endocannabinoids, which have modulatory effects on neurons and microglia, but are not synthetized by specific neurons. Their effects on microglia have been addressed in, for example,[START_REF] Calovi | Microglia and the Purinergic Signaling System[END_REF][START_REF] Cabral | Cannabinoid receptors in microglia of the central nervous system: immune functional relevance[END_REF].

ACKNOWLEDGMENTS:

We warmly thank Dr Catherine Béchade and Dr Luc Maroteaux for their corrections and comments. We thank the Cell and Tissue Imaging Facility of the Institut du Fer à Moulin, where the images presented in Figure 1 have been acquired and analyzed, and particularly Dr Irinopoulou for the 3D reconstruction on Imaris software.

FUNDINGS: This work has been supported in part by the Institut National de la Santé et de la Recherche Médicale, the Sorbonne Université Sciences, and by grants from Sorbonne Université-Pierre et Marie Curie University [Emergence-UPMC program 2011/2014], the Fondation pour la Recherche sur le Cerveau, the Fondation de France, the Fondation pour la Recherche Médicale [Equipe FRM DEQ2014039529] and the French Ministry of Research [Agence Nationale pour la Recherche ANR-17-CE16-0008 and Investissements d'Avenir programme "Bio-Psy Labex" ANR-11-IDEX-0004-02]. The authors are affiliated to a research group which is member of the Paris School of Neuroscience (ENP) and of the Bio-Psy Labex. A.R. is an Assistant Professor at Sorbonne Université, Paris, France. F.E. is a Ph.D. student affiliated with Sorbonne Université, Collège Doctoral, and is funded by the Bio-Psy Labex [ANR-11-IDEX-0004-02]. G.A. is a post-doctoral fellow funded by the Agence Nationale pour la Recherche [ANR-17-CE16-0008].

CONTRIBUTIONS:

All the authors outlined the subject and revised the manuscript. In addition, G.A. and A. R. wrote the introduction and the conclusion; G.A. wrote the paragraphs on dopamine and ACh, and prepared Figure 2 and the table; F.E. wrote the paragraphs on 5-HT; A.R. wrote the paragraphs on norepinephrine and histamine, prepared Figure 1 and the table, and supervised the whole process.

CONFLICTS OF INTEREST:

None.

REFERENCES:

[