Bertrand Bryche

Mikaël Le Bourhis

Patrice Congar

Claire Martin

Olivier Rampin

Dr Nicolas Meunier
email: nicolas.meunier@inra.fr

Endothelin impacts on olfactory processing in rats

Keywords: endothelin, carbenoxolone, electro-olfactogram, calcium signals, olfactory mucosa, behavioral tests OM, olfactory mucosa, OSN, olfactory sensory neurons, SCs, sustentacular cells, ET-1, endothelin, CBX, carbenoxolone

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

INTRODUCTION

Neuronal activity regulation was long thought to occur only in central structures, but it is now well established that it exists also in peripheral sensory neurons [START_REF] Lucero | Peripheral modulation of smell: fact or fiction?[END_REF]. Some of these regulations clearly involve glial cells of sensory organs [START_REF] Pack | Neuroglial modulation in peripheral sensory systems, The Neuroscientist : a review journal bringing neurobiology[END_REF]. The first step of odorant detection takes place in the olfactory mucosa (OM). In this neuroepithelium, olfactory sensory neurons (OSNs) are surrounded by glial-like sustentacular cells (SCs) forming a structural barrier. SCs also provide metabolic and trophic support to neurons and participate in the maintenance of ionic gradients in OSNs [START_REF] Getchell | Fine structural aspects of secretion and extrinsic innervation in the olfactory mucosa[END_REF]. Furthermore, part of the paracrine modulations of the OM implicates the SCs [START_REF] Dooley | Purinergic receptor antagonists inhibit odorant-mediated CREB phosphorylation in sustentacular cells of mouse olfactory epithelium[END_REF]. SCs express connexins 43 and 45, and are coupled by gap junctions [START_REF] Zhang | Expression of connexin 45 in the olfactory system[END_REF][START_REF] Vogalis | Electrical coupling in sustentacular cells of the mouse olfactory epithelium[END_REF]. Whereas the role of these gap junctions between SCs remains to be fully described [START_REF] Yu | The role of connexin 43 in mediating odor response[END_REF], the importance of gap junction communication has been broadly characterized in the central nervous system. Among others, they enable intercellular spread of electrical current, calcium wave propagation, and transmission of second messengers [START_REF] Herve | Gap-junction-mediated cell-to-cell communication[END_REF][START_REF] Nielsen | Gap junctions[END_REF]. Functions unrelated to intercellular communication such as cellular development or synaptic strength are now also well established for connexins [START_REF] Evans | Cell communication across gap junctions: a historical perspective and current developments[END_REF][START_REF] Pannasch | Connexin 30 sets synaptic strength by controlling astroglial synapse invasion[END_REF]. Endothelin (ET), one of the most potent vasoconstrictor peptides [START_REF] Yanagisawa | A novel potent vasoconstrictor peptide produced by vascular endothelial cells[END_REF], is also known to inhibit gap junctional communication very efficiently especially in astrocytes [START_REF] Blomstrand | Endothelins regulate astrocyte gap junctions in rat hippocampal slices[END_REF]. Three peptides of 21 amino acids named ET-1 to ET-3 are characterized, ET-1 being the most widely distributed [START_REF] Khodorova | Endothelin receptors and pain[END_REF]. These peptides are secreted as pro-peptides (Big-ETs), which are matured locally by peptidase endothelinconverting enzymes (ECEs) and then act autocrinally and paracrinally on two different G proteincoupled receptors, named ETA and ETB. The peptides along with their receptors and their converting enzymes are classically presented as the endothelin system. Based on primary cell cultures of the OM, we previously reported that ET-1 acutely triggers robust Ca 2+ transients in most cells. Using pharmacology, we observed that OSNs preferentially express ETB receptors while most non-neuronal cells preferentially express ETA receptors. The non-neuronal cells include mainly olfactory ensheathing cells (OECs) and SCs [START_REF] Gouadon | Endothelin evokes distinct calcium transients in neuronal and non-neuronal cells of rat olfactory mucosa primary cultures[END_REF]. In another study, we found that ET-1 acts as a neuroprotective peptide for the olfactory cells [START_REF] Laziz | Endothelin as a neuroprotective factor in the olfactory epithelium[END_REF]. While we have found that ET-1 could effectively uncouple gap junction communication, the protective effect did not appear to pass through gap junctions [START_REF] Bourhis | Endothelin uncouples gap junctions in sustentacular cells and olfactory ensheathing cells of the olfactory mucosa[END_REF].

Although ET-1 has been shown to mediate a rapid and reversible morphological plasticity of the OECs the possible role of ET-1 in SCs remains unknown. Therefore it is likely that ET-1 acts indirectly on the peripheral odor signal generated in the OM by uncoupling gap junction in SCs.

In the present study, we have focused on a potential effect of ET-1 on olfactory processing of young rats as our previous studies were performed on 10 day old pups [START_REF] Bourhis | Endothelin uncouples gap junctions in sustentacular cells and olfactory ensheathing cells of the olfactory mucosa[END_REF][START_REF] Francois | Daily oscillation of odorant detection in rat olfactory epithelium[END_REF]. We first confirmed ex-vivo with OM explants that ET-1, like a known gap junction uncoupling agent carbenoxolone (CBX), inhibits gap junctional permeability, limiting intercellular Ca 2+ wave propagation. Then, using electroolfactogram (EOG) recordings on hemi head, we found that ET-1 and CBX slowed down the repolarizing kinetics of the olfactory responses, without impacting their amplitudes. Finally, we tested the behavioral performances of rats in an odor-based orientation test. We confirmed the importance of ET-1 in olfactory processing but suggest that this effect does not rely solely on gap junction uncoupling as CBX application did not affect pup behavior.

Material and methods

Animals

Wistar male rat pups (Rattus norvegicus) were obtained from multiparous females, and housed in our local animal care facilities (Unité Expérimentale Animalerie Rongeurs (UEAR), Jouy-en-Josas,

Calcium imaging on OM explants

OM explants were prepared from Wistar male rat pups (post-natal day 10 to 13: P10-13) killed by decapitation. For the OM explants, upper skull bone, lower jaw, teeth and palate were removed and the explant placed in ice-cold artificial cerebrospinal fluid (ACSF) solution containing (mM): 124 NaCl, 5 KCl, 1.25 NaH2PO4, 2 MgSO4, 2 CaCl2, 26 NaHCO3 and 10 Glucose, saturated with 95% O2 and 5% CO2, pH 7.4, ~300 mOsmol/L). A paramedial sagittal section of the nose allowed harvesting of the medial septum that separates the two nasal cavities. OM attached to both sides of the septum was submerged in oxygenated ACSF until use.

Calcium-sensitive dye Oregon green (5 mM stock solution in DMSO) was mixed 1:1 with 20% Pluronic F-127 (Molecular Probes) and diluted in oxygenated ACSF to a loading solution of 20 µM. OM slices or OM explants attached to the septal wall were kept in the loading solution for 30-40 min, followed by a 10-min washing out period in oxygenated ACSF. OM explants were transferred, one at a time, to a home-made recording chamber and anchored by a customized net with the mucus layer facing up for explants. The recording chamber was gravity perfused continuously with oxygenated ACSF at a rate of ~2.5 ml/min, and placed under a x20 long-range water-immersion objective (XLUMPLFL20XW, NA, 0.95) of an upright microscope (BX51WI, Olympus, Rungis, France), equipped with differential interference contrast and an IR video camera (placed after a x40 magnification changer).

Fluorescence measurements were performed using a dedicated Xcellence RT real time live cell imaging system (Olympus, Soft Imaging Solutions-OSIS, Münster, Germany) consisting of a computercontrolled fast illumination MT20 system with a 150W Mercury-Xenon mixed gas short arc burner, and an cooled C9100-13 EM-CCD camera (Hamamatsu Photonics, Massy, France). Fluorescence images were collected at 535nm at 1 Hz, with short exposure times (10-100 ms) to minimize bleaching.

To evaluate gap junctions coupling between SCs, we generated electrical extracellular stimulation on OM explants. The electrical stimulation was delivered by a bipolar theta glass pipette filled with 1M NaCl solution (500mV; 500ms duration). The pipette was placed ~10 µm upon the tissue. A first stimulation was performed as a reference of signal propagation. It was followed by 30s of bath perfusion with either ACSF (n=22), ET-1 at 10 -7 M (n=32), CBX at 10 -4 M (n=20) or ATP at 10 -4 M (n=20).

This duration was long enough to fully exchange the bathing medium with the stimulating solution.

This bathing medium change was followed by 9.5 min of perfusion with ACSF allowing the recovery of the stimulated cells before applying a second identical electrical stimulation. The signal propagation was again compared between the same two ROIs. We measured the impact of the treatment on the gap junction communication as the percentage of propagation between second and first stimulation. Results are presented as mean ± standard error of the mean (SEM). For all offline analysis of calcium transients, images were background subtracted and changes in fluorescence were calculated relative to the averaged baseline (from 30s before stimulation) and expressed as %∆F/F ([(F-Fbaseline)/ Fbaseline]x100) using Xcellence RT software.

Electro-olfactogram (EOG) recording

EOG recordings were made from the olfactory mucosa of rat pups (P10-13) in an opened nasal cavity configuration as described earlier [START_REF] Francois | Olfactory epithelium changes in germfree mice[END_REF]. The hemi-head was placed in a recording chamber under an upright Olympus SZ51 stereo microscope (Olympus, Rungis, France) equipped with a low magnification objective (0.8 to 4x) and two MX-160 micromanipulators (Siskiyou, Inc., Grants Pass, OR, USA). The odor stimulation device was modified from Scott and Brierley [START_REF] Scott | A functional map in rat olfactory epithelium[END_REF]. The hemi-head was kept under a constant flow of humidified filtered air (~1000 ml/min) delivered through a 9 mm glass tube. This tube was positioned 2 cm from the epithelial surface. EOG responses were recorded until their amplitudes stabilized, (i.e. at least 3 successive responses displaying the same amplitude), and the last response was taken as the reference. One endoturbinate was treated by local application of a saline solution, while the other endoturbinate received the tested molecule (10 -3 M CBX (n=5) or 10 -6 M ET-1 (n=11)) through constant diffusion from a glass micropipette of ~10 µM diameter. The ionic composition of the saline solution was chosen to match as much as possible the mucus composition according to previous studies. Control and treated endoturbinates (IIb and III) were systematically alternated from one rat hemi-head to the other.

Responses were recorded on both endoturbinates simultaneously every 5min, for 20min following local treatment (Fig. 2B). Analysis were performed off-line using Clampfit 9.2 (Axon Instruments).

Peak amplitude, rise time (from 10% to 90%), as well as fast (from 100% to 80%) and slow (from 40% to 10%) decay slopes of EOG responses were measured. Since the EOG response kinetics highly correlate with the amplitude, the rise time and the two decay slopes were normalized to the corresponding response peak amplitude prior to statistical analysis. The amplitude and kinetics of the olfactory signals recorded following the drug applications were normalized to those of the control responses prior to this treatment. Results are presented as mean ± standard error of the mean (SEM).

Odorant driven orientation behavior

In order to evaluate the impact of gap junction communication on olfactory performances of rat pups (P10-12), we monitored their orientation toward nest odorants in a behavioral test as described previously [START_REF] Meunier | Using scalar products to refine the interpretative value of an orientation choice test[END_REF]. Tests were conducted between 10:00 and 11:00 a.m. on 12 litters of 10 to 12 Wistar rats (139 animals in total including male and female). For each litter, pups were separated from their mother 45 minutes prior to testing and marked for video-tracking purposes using low-odor markers (UniPosca, Mitsubishi Pencil Co. Ltd, Worcester, UK). Spots were made at the center of the head and at the center of the body (Fig. 3A). Prior to the test, pups from the same litter received 10 µL per nostril of the tested drug (10 -4 M CBX (n=27 from 6 litters) or 10 -7 M ET-1 (n=27 from 6 litters)) or saline as a control (n=28 or 29 respectively). 10 min after the nasal instillation, pups were introduced individually at the center of a closed custom-made box (LxWxH: 29.7 x 18.7 x 5.5cm3), with a continuous airflow incoming from two opposite compartments expelled by a small fan at the base of the box (~30L.min -1 ; i.e. ~10 box volume.min -1 ). One compartment contained sawdust collected from the home cage while the other contained only fresh sawdust. Videotaping was performed at 15 frames per second for 1min or until the animal tried to enter one of the compartments indicating a choice between the two odor sources. Pups treated with CBX or ET-1 were run alternatively in the test, while keeping exactly the same odor sources.

Videos were analyzed tracking both spots (head and body) using Kinovea freeware (http://www.kinovea.org/). We used the coordinates to calculate two unit vectors, one represented by the body dot to head dot (animal orientation vector) and one by the body dot to mother litter compartment (perfect orientation vector). For each frame, we calculated the difference of the scalar product of those vectors. A value of 2 indicates that both are aligned and of the same direction. In that case, the animal is perfectly orientated toward its mother smell while a scalar of -2 indicates the animal is perfectly orientated toward the fresh sawdust. A value of zero indicates no choice. If the animal chooses a compartment before the 1 min testing limit, the scalar value was assigned to 2 or -2 for the remaining frames according to the chosen compartment. The differences in product scalar values are then averaged for all frames to calculate a score of odor based orientation for each pups.

The score was normalized by the mean of the group treated with saline. We also monitored their average velocity and pups that did not move were excluded from the analysis (n=13/7/8 for saline, ET-1 and CBX treatment respectively). The pups not moving represented approximately 20 % of all animal tested (28 among 139). Results are presented as mean ± standard error of the mean (SEM).

Statistical analysis

Data were statistically analyzed using GraphPad Prism software (GraphPad software Inc., CA, USA).

We used one-way ANOVA for calcium imaging, two-way ANOVA for EOG analysis, and a Mann-Whitney test for behavior. When ANOVA indicated a significant effect, post-hoc test were used to determine individual differences between responses. We used Newman-Keuls multiple comparison of means test for one-way ANOVA, and Bonferroni multiple comparison of means for two-way ANOVA. A probability value of P<0.05 was used as an indication of significant differences.

RESULTS

ET-1 induces gap junction uncoupling of sustentacular cells ex vivo

Previously, using a primary culture of OM cells, we have demonstrated that SCs are sensitive to ET-1 [START_REF] Gouadon | Endothelin evokes distinct calcium transients in neuronal and non-neuronal cells of rat olfactory mucosa primary cultures[END_REF] and that ET-1 uncouples their gap junctions [START_REF] Bourhis | Endothelin uncouples gap junctions in sustentacular cells and olfactory ensheathing cells of the olfactory mucosa[END_REF]. In order to explore the biological relevance of this phenomenon in a preserved cellular environment of SCs, we used calcium imaging in OM explants to measure the impact of ET-1 treatment on the propagation of a calcium wave initiated by an electrical impulse (Fig. 1A). The propagation was evaluated by comparing the level of fluorescence induced near the stimulating electrode with the background fluorescence measured on a distant annular region. This relative propagation was analyzed on the same region, before and after application of either a saline solution (ACSF) or various pharmacological treatments. Results are presented as the relative propagation after treatment compared to the first stimulation used as a reference (Fig. 1B). A one-way ANOVA indicated significant differences among groups (P<0.0001). On average, signal propagation was reduced after the first electrical stimulation for saline solutiontreated cells (61% of remaining fluorescence), and post-hoc Newman-Keuls multiple comparison of the means indicated that this decrease was significantly enhanced after treatment with ET-1 and CBX (28.1 and 39.4% respectively) but not ATP (65.5%).

ET-1 and CBX application on the OE modulates odorant detection kinetics

In order to evaluate the impact of ET-1 and CBX on odorant detection, we performed electroolfactogram recordings (EOG) in an opened nasal cavity configuration (Fig. 2A). EOG signals arise from the summation of local depolarizations and repolarizations of OSNs during olfactory transduction [START_REF] Scott | The electroolfactogram: a review of its history and uses[END_REF]. Pilot experiments had shown that ET-1 application potentially modulates the kinetics of odorant responses of the OM for high concentration of odorants. We thus focused on the responses to isoamyl acetate diluted 1:10 in mineral oil. Simultaneous EOG recordings of the responses evoked by odorant stimulations were performed from the neighboring endoturbinates IIb and III for 20 min following treatment with either saline, 10 -6 M ET-1, or 10 -3 M CBX. Signals were normalized to the amplitude of the response prior to local treatment (Fig. 2B).

EOG signals are characterized by a fast decrease in amplitude reflecting the summation of olfactory transduction of OSNs near the recording electrode (Fig. 2C). Over time, the normalized EOG signal amplitude did not differ significantly between saline and treatment with ET-1 (F(1,80)=0.04; P=0.847) or CBX (F(1,32)=1.43; P=0.266) (Fig. 2D). After a few seconds following the odor stimulation, the signal returned to its initial value, reflecting the repolarization of OSNs. This repolarization is composed of a fast and slow part and only the latter appeared to be impacted by ET-1 and CBX treatment (Decay slope; Fig. 2C). The evolution over time of the slow component of the repolarizing phase was indeed significantly different from the saline treatment for both ET-1 (F(1,80)=8.81; P=0.018) and CBX (F(1,32)=16.51; P=0.0036) (Fig. 2D). These results indicate that ET-1 and CBX treatments delay the return of OSNs to the basal state of activity after odorant processing.

Odorant driven orientation behavior is impaired by ET-1 treatment but not by CBX

As the kinetic of OSN responses to an odorant was modulated by ET-1 and CBX, we next evaluated whether the same treatments would impact behavioral indices of olfactory detection. We used an orientation test for pups based on their innate attraction to maternal odors. At 12 days of age, rat pups are still blind and thus rely mainly on olfactory cues for orientation. In this test, we calculated the difference between two scalar products of unit vectors to score the orientation of the pup during the test (Fig. 3A). The difference in scalar product ranges from positive to negative values, respectively indicating a direct or opposite orientation of the pup toward the maternal odor source (Fig. 3B). We measured an orientation score based on the mean of the difference in scalar product for each frames. A Mann-Whitney test indicated that ET-1 treatment significantly lowered the orientation score of the pups compared to saline (P=0.005) while the CBX treatment did not (P=0.78; Fig. 3C). The velocity was not statistically different between groups (2.87±0.44 vs 3.06±0.55 cm/s for saline and ET-1 group respectively; 3.03±0.35 vs 2.99±0.37 cm/s for saline and CBX group respectively).

Discussion

We have previously established that ET receptors were highly expressed in the OM [START_REF] Bourhis | Endothelin uncouples gap junctions in sustentacular cells and olfactory ensheathing cells of the olfactory mucosa[END_REF]. We have also found that ET-1 could uncouple gap junction communication in OM primary culture cells, with no clear link to its anti-apoptotic effect [START_REF] Laziz | Endothelin as a neuroprotective factor in the olfactory epithelium[END_REF]. In the present study, we investigated the impact of gap junction uncoupling by ET-1 on odorant detection.

In order to confirm the gap junction uncoupling effect of ET-1 on SCs in a preserved preparation, we first performed ex vivo calcium imaging experiments on explant of OM. In this preparation, all the apical cell bodies that can be visualized for imaging are SCs. We observed that both ET-1 and CBX significantly reduced the calcium wave propagation initiated locally by electrical stimulation. This observation is consistent with our previous study showing that ET-1 and CBX reduced gap junction coupling between SCs in primary culture. We next examined whether local application of ET-1 and CBX on the surface of the OM would impact odorant detection. This detection is initiated in the OSN cilia where odorants binding to olfactory receptors induces changes in the activation state of those GPCRs, leading to modulation of the membrane potential [START_REF] Dibattista | The long tale of the calcium activated Cl-Channels in olfactory transduction[END_REF]. EOG allows the recording of the summation of responses to odorant stimulation for thousands of OSNs [START_REF] Scott | The electroolfactogram: a review of its history and uses[END_REF]. We observed that ET-1 and CBX did not impact the amplitude of the odorant-induced EOG signal but slowed its recovery.

The EOG amplitude signal is related to the olfactory receptor potential and the kinetic of the repolarization could be linked to the ionic composition of the mucus as shown before [START_REF] Griff | A selective PMCA inhibitor does not prolong the electroolfactogram in mouse[END_REF]. Indeed, the olfactory signal transduction consists of an inward current of sodium and calcium which in turn opens chloride channel allowing a chloride efflux. All those ionic movements impact the local ionic concentration surrounding OSN cilia and their concentration has to be restored in order to return to the basal state of OSN activity. Alteration of mucus ionic composition is known to impact the EOG signal [START_REF] Takagi | The role of sodium and potassium ions in the generation of the electro-olfactogram[END_REF]. SCs may play a key role in maintaining the ionic gradient required to fulfill the olfactory transduction, and uncoupling their gap junction could thus impact the restoration of mucus composition. Gap junctions are also present in OSN including Cx57 [START_REF] Zhang | Expression of connexin 57 in the olfactory epithelium and olfactory bulb[END_REF], thus the observed effect could be linked to a direct action of ET-1 and CBX on OSNs. However this seems unlikely, as EOG reflects the olfactory transduction in OSN cilia where gap junctions are absent. This would also be consistent with another study performed on the salamander Necturus maculosus, indicating a role for the coupling of OSN in cellular dynamics rather than olfactory transduction [START_REF] Delay | Coupling between sensory neurons in the olfactory epithelium[END_REF]. Our result contrasts with a study in mice expressing a dominant negative form of Cx43 in OSNs [START_REF] Zhang | Gap junctions in olfactory neurons modulate olfactory sensitivity[END_REF]. In those mice, the response to odorants recorded by EOG and calcium imaging were reduced. Similarly the authors observed that CBX application induced a decrease in response to odorant stimulation with calcium imaging [START_REF] Yu | The role of connexin 43 in mediating odor response[END_REF]. The main differences with our study may be the animal age (10 day old rats versus 8-16 weeks mice) and the protocol for OM preparation. We used aerial EOG while the previous results were obtained with the OM immersed in saline. Thus, our experimental design preserves the mucus originating from the OM as much as possible, which could explain the difference between studies. Indeed, as the mucus layer is still present around OSN cilia, so are most of the detoxifying enzyme that may limit the action of ET-1 and CBX [START_REF] Heydel | Odorant-binding proteins and xenobiotic metabolizing enzymes: implications in olfactory perireceptor events[END_REF]. Thus an impact of gap junction uncoupling agent on the mucus ionic composition may be seen with an aerial preparation but overlooked with an immersed preparation.

We next examined how the observed change in odorant detection could affect the animal ability to smell. The kinetic study of ET-1 action on OSN repolarization after odor stimulation indicates that ET-1 was most effective 10 min after treatment. Such delay matches the time window used to explore the impact of ET-1 on calcium wave propagation induced by electrical stimulation on OM explants.

We thus used the same treatment time to study the impact on ET-1 nasal instillation on olfactory performances. Using a two odorant choice test, we found that ET-1 but not CBX intranasal instillation reduced the olfactory performances of rat pups. As CBX and ET-1 OM application have similar effects on the gap junction communication and odorant transduction kinetic, this indicates that in our study, the slowed repolarization of OSNs has no major impact on the ability of an animal to smell. These results contrast with the work on OMP KO mice where OSN repolarization is impaired along with deficits in olfactory performances [START_REF] Reisert | Olfactory marker protein modulates the cAMP kinetics of the odour-induced response in cilia of mouse olfactory receptor neurons[END_REF]. However, OMP has been also described to be essential for olfactory bulb organization [START_REF] Albeanu | Olfactory marker protein (OMP) regulates formation and refinement of the olfactory glomerular map[END_REF]. The difference between ET-1 and CBX treatment on odor based behavior of pups remains thus to be explored. A simple explanation would be that the steroid-like structure of CBX may be more sensitive to degradation by detoxifying enzymes of the mucus and thus impact less the odorant detection than ET-1 in vivo [START_REF] Heydel | Odorant-binding proteins and xenobiotic metabolizing enzymes: implications in olfactory perireceptor events[END_REF]. Another explanation could be that ET-1 have targets additional to CBX on the OM. Endothelin has first been characterized for its vasoconstrictor effect [START_REF] Yanagisawa | A novel potent vasoconstrictor peptide produced by vascular endothelial cells[END_REF] and one could expect to observe a decrease of olfactory performances due to reduced blood flow in the OM treated with ET-1. This hypothesis is unlikely as other vasoconstrictor messengers like epinephrine are known to improve the ability to smell [START_REF] Escanilla | Noradrenergic modulation of behavioral odor detection and discrimination thresholds in the olfactory bulb[END_REF] and αadrenergic receptor agonists are classically used as nasal decongestants without impairment of olfactory performances [START_REF] Johnson | The pharmacology of alpha-adrenergic decongestants[END_REF][START_REF] Jung | Influence of intranasal epinephrine and lidocaine spray on olfactory function tests in healthy human subjects, Otolaryngology--head and neck surgery[END_REF]. A more plausible possibility is that ET-1 not only acts on SCs but also on OSNs. Indeed, gap junction uncoupling may act both on the receptor potential through action on SCs and afterward the calcium wave in the OSNs cell body. ET-1 could act on OSN through modulation of Cx43 phosphorylation as described by [START_REF] Yu | The role of connexin 43 in mediating odor response[END_REF]. ET-1 could also act more deeply in the OM as peptides can cross the OM and reach the central nervous system [START_REF] Crowe | Mechanism of intranasal drug delivery directly to the brain[END_REF]. It could then act on the OECs surrounding the axon bundles of OSNs [START_REF] Bourhis | Endothelin uncouples gap junctions in sustentacular cells and olfactory ensheathing cells of the olfactory mucosa[END_REF]. Gap junction uncoupling of OECs suppress the propagation of calcium wave between those cells [START_REF] Stavermann | Calcium-induced calcium release and gap junctions mediate large-scale calcium waves in olfactory ensheathing cells in situ[END_REF] potentially modulating the transmission of the information from the OM to the olfactory bulb [START_REF] Thyssen | Ectopic vesicular neurotransmitter release along sensory axons mediates neurovascular coupling via glial calcium signaling[END_REF].

Further studies are required to investigate whether our results also apply to adult rats. Indeed, in 10 day old rats the nervous system is immature and GAP junction component expression is known to change according to the development stage [START_REF] Dermietzel | Differential expression of three gap junction proteins in developing and mature brain tissues[END_REF]. Our study also leaves many questions unanswered.

Under what physiological conditions are ET levels modulated in the OM? Would food odors be similarly affected as home cage odors? Further studies are required to explore those questions.

France) in 12

 12 h light, 12 h dark cycles. Mothers were fed water and standard chow ad libitum and bedding was changed weekly. All animal experiments were conducted in accordance with the European Communities Council Directive 2010/63/EU on the protection of animals used for scientific purposes, and approved by our French Local Ethics Committee (Comethea, CNREEA # 45; validated protocol #12-058). All efforts were made to minimize the number and suffering of the rats used. PC and NM hold the authorization for performing experiments in animals, including the animals experiments conducted in the present study, provided by Préfecture des Yvelines (France), according to French and European laws (agreements #78-154).

  Odor stimulations were performed by blowing air puffs (200 ms, 200 ml/min) through an exchangeable Pasteur pipette enclosed in the glass tube containing a filter paper impregnated with 20 µL of odorant (Sigma Aldrich, Saint-Quentin Fallavier, France). Recordings were made with glass micropipettes of 4-5MΩ. Simultaneous EOG recordings of the responses evoked by isoamyl acetate (1:10 in mineral oil) were recorded from endoturbinates IIb and III. The recording electrodes were placed in the center of these endoturbinates. This position gave robust, reproducible and long-lasting EOG recordings. Stimulation with only mineral oil always produced signals of less than 1 mV amplitude.

Figure 1 :

 1 Figure 1: ET-1 application decreased the propagation of a calcium wave in an OM explant similarly as

Figure 2 :

 2 Figure 2: Kinetic of olfactory transduction is slowed down by ET-1 and CBX treatments. (A) Schematic

Figure 3 :

 3 Figure 3: Orientation based on olfactory cues is impaired by ET-1 but not CBX treatment. (A) Aerial

Acknowledgment

This work was funded by the Institut National de la Recherche Agronomique (INRA). We would like to thank Birte Nielsen and Vincent Bombail for their help in improving the manuscript, Patrice Dahirel and Aurelien Raynaud for animal care and treatments.

Conflict of interests

The authors declare no conflict of interests.


Marie-Hélène Mammez

Raphaël Vallon

Frédéric Polak

Bertrand Parvitte

Virginie Zéninari

Design and implementation of a heliostat for atmospheric spectroscopy

Keywords: Heliostat, coordinate system, servo control, FTIR, atmospheric spectroscopy

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.

Introduction: context and state-of-art

Optical laser spectrometry is a tool based on the Beer-Lambert law and widely used for atmospheric studies, either in situ -for example CEAS (Cavity Enhanced Absorption Spectroscopy) [START_REF] Gagliardi | Cavity-Enhanced Spectroscopy and Sensing[END_REF] and photoacoustic spectroscopy [START_REF] Michaelian | Photoacoustic IR spectroscopy instrumentation, applications and data analysis[END_REF] or remotely -for example LIDAR (Light Detection And Ranging) [3] and heterodyne spectrometry [START_REF] Parvitte | Infrared laser heterodyne systems[END_REF]. The heterodyne spectroscopy is a passive groundbased technique particularly used for atmospherical [START_REF] Parvitte | Preliminary results of heterodyne detection with quantum-cascade lasers in the 9 µm region[END_REF][START_REF] Weidmann | Ground-based prototype quantum cascade laser heterodyne radiometer for atmospheric studies[END_REF]

and astrophysical

Email address: virginie.zeninari@univ-reims.fr (Virginie Zéninari) studies [START_REF] Sonnabend | Tuneable heterodyne infrared spectrometer for atmospheric and astronomical studies[END_REF]. In the atmospherical case, a part of the sunlight must be collected by a heliostat and superimposed to a local oscillator beam in order to create a beat note signal.

There are mainly two types of heliostat structures which enable to collect the solar beam for its spectrometric analysis: the systems with equatorial mounts [START_REF] Cageao | High-resolution fourier-transform ultravioletvisible spectrometer for the measurement of atmospheric trace species: Application to OH[END_REF] and the systems with altazimuthal mounts [START_REF] Delahaigue | Design of a sun tracker for a laser heterodyne spectrometer[END_REF]. Equatorial mounts enable a precise Sun tracking but have the disadvantage of transmitting a mobile output beam which is unsuitable with the heterodyne measurement (no possible superposition with the local oscillator beam). On the contrary, the altazimuthal mounts lead to a fixed output beam. A commercial version (Bruker A547) of this kind of instrument is available and has been successfully used [START_REF] Geibel | A new fully automated FTIR system for total column measurements of greenhouse gases[END_REF][START_REF] Té | The Fourier transform spectrometer of the Université Pierre et Marie Curie QualAir platform[END_REF].

Nonetheless the optical path length between tracker head and quadrant detector must not exceed 5 m [START_REF]SOLAR TRACKER A 547 accessory manual[END_REF].

If the spectrometer is located far from the roof, a non-commercial altazimuthal structure is preferable. Among those systems, the Sun tracking can be carried out either by calculation or by a servo loop. Contrary to trackers with a servo loop, the computed tracking can't take into account the atmospheric perturbations which cause unpredictable beam movements [START_REF] Ciddor | Refractive index of air: New equations for the visible and near infrared[END_REF].

The servo control is often carried out by generating an error signal from a quadrant photodiode [START_REF] Merlaud | Equations for solar tracking[END_REF] or a camera [START_REF] Gisi | Camtracker: a new camera controlled high precision solar tracker system for ftir-spectrometers[END_REF]. A quadrant photodiode delivers an error signal which depends on the power distribution and the shape of the solar beam. A camera is the best system to generate the error signal because it enables to consider the form of the tracked object, for example in the case of the Moon. But it requires to use a heavy image processing to calculate the Sun position and generate an error signal. In those two cases, the raw error signal isn't efficient to correct the rotation of the instrument: first it must be compared with the approximate calculation of the solar position via a matrix processing [START_REF] Jin | A sun tracking system design for a large dish solar concentrator[END_REF][START_REF] Chong | General formula for on-axis sun-tracking system and its application in improving tracking accuracy of solar collector[END_REF][START_REF] Chong | Integration of an on-axis general sun-tracking formula in the algorithm of an open-loop sun-tracking system[END_REF][START_REF] Chong | General Formula for On-Axis Sun-Tracking System[END_REF]. As a result, the servo-controlled tracking becomes delicate close to the zenith because the azimuth angle isn't discriminative there and can take any value. Therefore with this servo-control method the heliostat becomes uncontrollable close to the zenith. Existing instruments usually bypass this problem by temporarily disengaging the servo loop for the azimuthal rotation [START_REF] Merlaud | Equations for solar tracking[END_REF]. Thus monitoring is less effective in the middle of the day. To avoid the complete stop of the azimuthal servo-control, its parameters PID (proportional -integral -derivative) could be adapted according to the time. Recently, Volkov et al. [START_REF] Volkov | Optical model and calibration of a sun tracker[END_REF] defined an optimization method which enables to solve the stability loss near the zenith.

The GSMA is currently developing a heterodyne spectrometer using quantum cascade lasers as local oscillator. In vue to use our heterodyne spectrometer for atmospheric studies, we have developed a heliostat. We report here the development of a servo-controlled instrument adapted to the constraints imposed by infrared heterodyne spectrometry and the layout of the places (need to obtain a fixed output beam, 10 m distance between the heliostat and the spectrometer).

It's a servo-controlled instrument for which the error signal is produced by a lateral effect photodiode (LEP) and is independent of the shape, size or power distribution of the incident beam. The servo-control method that we present is a bit different than Volkov et al.'s [START_REF] Volkov | Optical model and calibration of a sun tracker[END_REF] but enables to track efficiently the Sun whatever the time, without the complexity of an adaptive PID and without the stability loss near the zenith.

Experimental set-up and operating principle

Optical set-up

The heliostat or suntracker that we built is composed of two plane mirrors M 1 and M 2 with elliptical section (see Figure 1). They are inclined at 45 • relative to the vertical of the location. M 2 adjusts the zenithal angle θ 2 and the set {M 1 + M 2 } ensures the θ 1 azimuthal rotation. The mirrors are connected to motorized rotation stages. It is split into two paths by a BaF 2 beamsplitter (reflection of 4% per side). The reflected beam is focused on a Lateral Effect Photodiode (LEP, PDP90A -Thorlabs), sensitive to visible radiation, and which enables the servo-control suntracking. The transmitted beam is redirected by two plane mirrors towards the spectrometer through a skylight. This device enables to collect a total power beam of ∼ 390 mW by full sunlight. The heliostat is protected by a double-walled housing to limit parasitic illumination of the LEP and disturbances due to wind. It is surmounted by an electro-pneumatic opening lid. Ventilation and heating system have been added to minimize moisture inside the box and avoid deterioration of optics.

For safety reasons, the closing of the system has been designed so as to minimize the need to access the roof. The tilting lid is caused by a pneumatic piston whose control is provided from the experiment room. The lid control program communicates with the rotation stages and with a reading program of a weather probe (WTX510 -Vaisala). It enables to protect the device in inclement weather by an automatic stop and closure of the tracker.

Field of view and angular resolution

Accurate solar tracking is a critical component of many quantitative approaches to remote sounding of our atmosphere. The retrieval of trace gas columns from an observed solar spectrum requires precise knowledge of the instruments line-of-sight (LOS) at the time of observation. The required stability for this kind of applications is typically better than ±0.01 • (±36 arcsec) [START_REF] Franklin | Solar Absorption Spectroscopy at the Dalhousie Atmospheric Observatory[END_REF].

For measurements with FTIR specrometers [START_REF] Chen | Differential column measurements using compact solar-tracking spectrometers[END_REF] and for heterodyne measurements it is also necessary to maintain a constant light intensity. So it must be ensured that the target point is always the same, for example the center of the Sun. Thus the whole image must be perceived on the LEP for a good pointing. This is only possible if the apparent diameter of the Sun (α = 9 mrad) is smaller than the field of view (FOV) of the device, as it is shown on Figure 2. Otherwise a shift smaller than the apparent diameter of the Sun will not be perceived by the servo system (Figure 2). In practice, the FOV is limited by the smallest field of view of the components of the heliostat [START_REF] Merlaud | Equations for solar tracking[END_REF].

F OV = min (F OV 1 , F OV 2 ) > 9 mrad (1)
where

F OV 1 = arctan φ D f (2)
F OV 2 = arctan φ T L 0 (3)
and with:

• F OV 1 : the field of view of the LEP

• F OV 2 : the field of view of the beamsplitter

• φ D = 10 mm : the diameter of the LEP,

• f = 12.5 cm : the focal length of the lens which focus the beam on the LEP,

• φ T : the aperture diameter of the heliostat (i.e. the aperture diameter of the azimuthal rotation stage),

• L 0 : the distance between the suntracker and the servo system.

The stage selected for the zenithal rotation has a resolution of 2.1 µrad for a 2 cm-aperture (M-060.DG -PI -Physik Instrumente). The stage of the azimuthal rotation has a resolution of 0.96 µrad for a 4.5 cm-aperture (M-062.DG -PI). As we can see on Figure 2, the field of view of the instrument is limitted by the aperture of the azimuthal stage. That means the distance L 0 must be smaller than 5 m. However, the spectrometer is placed in the experimental room at L ≈ 10 m from the roof. So the servo system must be set up on the roof near the stages for a sufficient field of view (cf equation 1).

Although the heliostat is designed to operate with a servo loop, the use of a LEP for generating the error signal requires to roughly know the position of the Sun in order to illuminate the LEP and get a starting error signal. Consequently the heliostat must first be programmed to calculate and reach an adequate starting position. Then, the servo loop enables to center the beam on the LEP.

The zenithal and azimuthal angles are considered correct when the image of the Sun coincides with the LEP center.

Calculation of solar positions

In the case of a perfectly oriented instrument, the suntracking can be done by calculating its positions during the day. A Matlab program using the algorithm of Jean Meeus is used [START_REF] Meeus | Astronomical algorithms[END_REF][START_REF] Reda | Solar position algorithm for solar radiation applications[END_REF]. Zenithal and azimuthal angles of the Sun are obtained in a topocentric coordinate system. The calculation is made from the geographical coordinates of the tracker, the date, time and offset relative to Coordinated Universal Time (UTC). The zenithal angle θ 2 is measured relative to the local vertical, that is to say the altitude alt = 90 • -θ 2 . The azimuthal angle θ 1 is measured in the tangent plane to the Earth from North and eastward (Figure 3). This reference is named topocentric coodinate system and is sketched on Figure 3.

The algorithm calculates the position of the Sun between -2000 and 6000 years, with an uncertainty of 0.0003 • or 5.2 µrad. This precision is degraded for large zenithal angles because of atmospheric refraction. It is also limited by the irregular variations of the Earth rotation. In addition, due to the imperfect orientation of the heliostat, the coordinate system considered in the calculation doesn't coincide with the real coordinate system of the physical instrument. This difference corresponds to an angular shift between the two coordinate systems and leads to an increasing of the measured error. A total error of ± 2.2 • is measured on the zenithal angle; and a total error of ± 8.1 • is measured on the azimuthal angle. These results are reported on Figure 4 (a). Under these conditions the LEP of the servo control is not illuminated.

Generally, Euler angles are used to correct the orientation error of the instrument [START_REF] Volkov | Optical model and calibration of a sun tracker[END_REF] as they represent the angular shift between the theoretical coordinate system and the real one. A correction with this method enables to reduce the error of 27% for zenithal angles, and 88% for azimuths. But in our case it was not enougth to get a beam down our skylight. That's why we preferred to realise a calibration of our setup, which is made by fitting the curve of the calculated positions compared to the measured positions. The maximum error is then reduced to ± 0.9 • for zenithal angles and ± 0.2 • for azimuths, as it is shown on Figure 4 (b). So, the calculation accuracy is improved by 59% for zenithal angles and by 99% for azimuths. We can note that the calibration method is here twice better than Euler angles method to correct zenithal error. Moreover the beam reaches the bottom of the skylight.

After the correction on the calculated Sun position, the beam shift measured down the skylight is 16 cm depending on the zenithal rotation and 3.5 cm depending on the azimuthal rotation. This shift is not a problem here because the calculation is only used to illuminate the LEP placed on the roof (up the skylight): the beam is centered on the LEP in a second step by the servo loop.

More details about the calculation correction are reported in [START_REF] Mammez | Détection hétérodyne de molécules d'intérêt atmosphérique à l'aide de lasers à cascade quantique[END_REF].

Servo loop

The servo system consists in generating an error signal for correcting the rotation of the mirrors through a feedback loop. This is usually associated with a PID to optimize the response time. Generating the error signal can typically be made by a quadrant photodiode [START_REF] Merlaud | Equations for solar tracking[END_REF][START_REF] Lee | Sun tracking systems: a review[END_REF][START_REF] Roth | Design and construction of a system for sun-tracking[END_REF] or a camera [START_REF] Gisi | Camtracker: a new camera controlled high precision solar tracker system for ftir-spectrometers[END_REF]. where the servo control is turned off and each stage is manually moved to see the movement induced on the beam.

Selected stages operate in a closed loop, that is to say, our program indicates the target-angle to the controller (C-863 -PI) which transmits the movement command to the stage and checks whether the displacement occurs (unlike the open loop stages that only transmit order without testing execution). Moreover, the stages do not have analog input and are chained on a single controller (series circuit). Consequently the error signal from the LEP can not be directly transmitted to a stage. It must be processed to determine the angular correction to be imposed. This correction is then digitally transmitted by the controller to the stage. Therefore, the traditional setting of a PID can not be implemented.

The servo loop is mainly limited by the error signals coupling. One solution is to calculate an approximate sunposition to know the sign of the correction to apply and then rotating step by step the azimuthal and zenithal stages to optimize the error signal [START_REF] Merlaud | Equations for solar tracking[END_REF]. Using such a method with altazimuthal geometry leads to an anarchic response of the system approaching the zenith (θ 2 = 0). At this time of the day, the error signal is not strictly discriminative since the Sun can be found whatever the value of the azimuthal angle. This problem is often bypassed by disengaging the servo loop of the azimuthal rotation when the Sun is near the zenith. Another solution consists in using an adaptive PID. None of these operations is required with the servo method developed in this work and detailed below.

As it is shown on Figure 5, the rotational movement of each stage results in a curvilinear motion on the LEP assimilable with a straight line around its center.

Each of the two straight lines represents the coordinate axis of the associated stage. So the altazenithal coordinate system can be projected in the coordinate system of the LEP, as it is shown on Figure 6. As the rotation stages axis are perpendicular to each other, the zenithal axis is always perpendicular to the azimuthal one; and the modeling of the altazimuthal coordinate system can be simplified by focusing on the movement of azimuthal axis. During the day, the projection of the altazimuthal coordinate system (coordinate sytem of the rotation stages) turns with an angle ϕ relative to the coordinate system of the LEP, as displayed in Figure 6. This movement is independent of the path of the Sun but only depends on the mecanical position imposed to the azimuthal stage. Indeed, the rotation stages of the heliostat and the LEP of the servo are fixed on a common mechanical structure, so the correspondence between the two systems can't be modified.

The angle ϕ was recorded during one day. Its expression is established depending on the azimuth by fitting the curve on Figure 7 (a). The fit is calculated in such a way as to impose a strictly increasing evolution to ϕ and so to prevent degeneracy of the azimuth near the zenith (θ 2 = 0). At this time of the day, just one azimuthal value is now allowed, and there is no anarchic response of the tracker. During the tracking, ϕ is estimated from the azimuthal angle measurement. Then the X and Y values measured on the LEP are projected onto zenithal and azimuthal axes noted x and y using equation 4. This process is described on Figure 7 (b). Therefore, two new error signals independent from each other are generated (one for each stage). Azimuthal and zenithal angles can be then adjusted in decoupled and simultaneous steps.

     x = -X cos ϕ + Y sin ϕ y = X sin ϕ + Y cos ϕ (4)
The servo loop effect on the suntracking is shown on Figure 8 (a) and Figure 9 (a). The observed behavior is consistent with that of a conventional servo loop using PID. On the roof, the amplitude of displacement of the solar spot on the surface of the LEP after focusing (BaF 2 lens with a 12.5 cm focal length) is 12 µm along the X axis and 7.4 µm along the Y axis (Figure 8 (b) and Figure 9 (b)).

Although this servo loop is not programmed with a conventional PID, it still enables to replicate its behavior. The angular correction is proportional to the error signal which is similar to a P setting. The I integration aspect is connected to the repetition rate of the servo loop: the loop time is imposed at a maximum duration of 850 ms. Only the derivation D setting is not taken into account: it is not essential because the Sun movement is slow (73 µrad/s).

Down the skylight, a part of the beam is focused on a second LEP using a second BaF 2 lens with a 50 mm focal length to quantify the beam stability at L = 10 m from the heliostat. As it is displayed on Figure 8 (c) and Figure 9 (c), the amplitude of displacement of the solar spot on the surface of this second LEP is 1.1 × 10 -3 mm on both X and Y directions. This is associated with an experimental total angular error of θ = 2.2 × 10 -5 rad (θ = 1.3 × 10 -3• ). This error is twice (Delahaigue et al. [START_REF] Delahaigue | Design of a sun tracker for a laser heterodyne spectrometer[END_REF]) or three times (Gisi et al. [START_REF] Gisi | Camtracker: a new camera controlled high precision solar tracker system for ftir-spectrometers[END_REF]) better than in other systems where the distance L is respectively three and four times smaller. So in input of this second lens, the maximum shift (δ) of the solar beam is δ = L × θ = 10 -4 µm. Figure 8 (c) and Figure 9 (c) also show the ability of the servo loop to stabilize the direction of the output beam and to compensate the effects of mechanical/thermal/alignment defects of the heliostat.

The servo control described here can only exist if the LEP is illuminated: it depends strongly on the weather conditions (clouds). Thus the control program provides for the switchover from the servo mode to a calculated mode (based on the ephemeris) when the intensity measured by the LEP is less than a threshold value. Similarly, switching from the calculated mode to the servo mode is performed automatically when the intensity is sufficient anew.

We have compared our servo loop efficiancy to Volkov et al.'s method [START_REF] Volkov | Optical model and calibration of a sun tracker[END_REF].

They have chosen to determine theoretically the correction to apply on Euler angles to rectify the correspondence between the theoretical and real coordinate systems. They obtained an experimental stability of 0.01 • . In our case, we have calibrated the instrument by projecting the axis of the rotation stages on that of the servo LEP to get an angular error 10 times smaller (2.2 × 10 -5 rad).

Atmospheric spectra

Down the skylight, solar beam reaches a 11 cm diameter. A 3" diameter part of the beam is taken and its size is reduced by a factor 4.5 by the use two off-axis parabolic mirrors. This beam shaping is suitable for direct injection, ultimately, in a heterodyne spectrometer currently developed at our laboratory from optical diameter of 1".

In order to test the heliostat performances for spectroscopy, the system is also adapted to inject the solar beam in a Fourier Transform Infrared spectrometer (FTIR, Jasco 6300) which is located at the other end of the room. There are 4 m between the skylight output and the FTIR spectrometer. So an additional optical system consisting of two spherical confocal mirrors (f 1 = 100 mm et

f 2 = 200 mm
) is added in order to adapt the beam to the spectrometer entry:

the beam size is divided by a factor two. Thus unlike all other instruments presented in the literature (for which the spectrometer should be placed just next to their servo control captor), there are ≈14 m from our servo system up to our FTIR spectrometer. Our heliostat has enabled us to record atmospheric spectra during 10 h without interruption (no optical realignment, no anarchic response at the zenith), and with a 0.15 cm -1 resolution. One of those spectra is presented on the top of Figure 10, and compared with simulations in the 900 cm -1 -1200 cm -1 range.

The simulated spectra were obtained with the 4A/OP radiative transfer calculation software using several database notably the GEISA 2011 database [START_REF] Scott | A fast line-by-line method for atmospheric absorption computations: The automatized atmospheric absorption atlas[END_REF][START_REF] Chaumat | 4A/OP Reference documentation[END_REF]. It has been used to estimate the radiance at ground level. However, it may differ slightly from experimental spectrum because 4A/OP provides two default types of profiles (pressure, temperature, main molecules concentration), corresponding to tropical and polar environments. None of them are really suitable for mid-latitudes. We have chosen to use a polar atmosphere profile which is a priori closer to real conditions, especially for the amount of water vapor.

There is a good agreement between the recorded spectrum and the simulation reported in the middle of Figure 10. But we can note that the calculated spectrum has deeper transitions than the recorded one. This is explained by the non-inclusion of the apparatus function of the spectrometer. Convoluting the calculated spectrum with a 0.15 cm -1 -width-Gaussian function, the spectrum is in better agreement with the measure, as it is shown at the bottom of Figure 10.

We compare also the measured spectrum with the simulated absorption spectra of different atmospheric molecules (4A/OP software): ozone, water vapor, carbon dioxide and nitrous oxide in the 900 cm -1 -1200 cm -1 range (Figure 11). Their respective contributions to the total absorption can be identified. Indeed one can distinguish in particular the characteristic shape of the absorption band of O 3 around 1050 cm -1 , the contribution of water lines in 900 cm -1 -1000 cm -1 and 1100 cm -1 -1200 cm -1 ranges, the two absorption bands of CO 2 between 900 cm -1 and 1100 cm -1 , and the band of N 2 O between 1150 cm -1 and 1200 cm -1 . The agreement between this experimental spectrum and the simulations confirms the relevance of the heliostat for spectrometry analysis.

Conclusion

The heliostat presented here operates without loss of control of the servo loop as it approaches the zenith. It enables to track the Sun for a whole day regardless of the time of year (measurements made made at differents times of the year).

The servo-control method developed in this work is effective enough to collect a solar beam and to stabilize it with an total angular error of 2.2 × 10 -5 rad.

According to our knowledge, this stability is better than the other suntrackers described in the literature. Unlike other published systems, thanks to this great stability, the instrument that we designed and implemented can be located at a great distance from the spectrometer (the beam propagates in a 14 m-free space). The relevance of the instrument for spectroscopic measurements was validated by the record of atmospheric spectra by FTIR spectrometry which are in agreement with the simulation.

Figure 1 :

 1 Figure 1: Optical set-up. A fraction of the sunlignt is taken by M 2 mirror and transmited by the M 1 mirror through a hole in the azimuthal stage (4.5 cm-aperture). The transmitted beam is divided in two parts: the first is directed towards the spectrometer via the skylight; the second is focused on a lateral effect photodiode for the servo control. The whole setup is placed on the roof of the laboratory.

Figure 2 :

 2 Figure 2: Impact of the field of view of the heliostat on the servo loop: if F OV > α the control accuracy is limited by the sensor resolution. In our case, it requires placing the servo device on the roof because of the too small aperture of the azimuthal stage.

  A LEP (PDP90A -Thorlabs) which has an intermediate comportment was chosen. It enables to determine the position of the Sun's image on its surface, regardless of its form or power distribution, and avoiding the constraints related to image processing. The calculation presentef in section 3 enables to illuminate the LEP which then generates two error signals (voltages) indicating the differences in X and Y between the beam position and the center of the LEP. Those signals are coupled signals which depend on movements of the two stages. The X and Y error signals produced by the LEP are correlated and both of them depend on angles imposed on the two rotation stages. Therefore the error signals can not be used independently of each other. Both of them contribute to the correction of the positions of the two stages. This phenomenon is highlighted in Figure 5

Figure 3 :

 3 Figure 3: (Top) The topocentric coordinate system is built with the location of the heliostat as the origin point of the reference, the local vertical and the tangent plane to the Earth. (Bottom) The zenithal angle θ 2 is measured from the local vertical. The azimuth θ 1 is measured in the perpendicular plane from the North to the East.

Figure 4 :

 4 Figure 4: Calculated mode operation (without servo control). (a) Error measured before correction. (b) Error measured after correction. The correction of the calculation by the calibration improves the error |measured-calculated| of the stages positions by 59% for zenithal angles and by 99% for azimuths.

Figure 5 :

 5 Figure 5: Evolution of the position (X, Y) of the beam on the LEP highlighting the coupling of error signals. The servo control is turned off and the stages are manually turned to show the movement induced on the beam by each of them. X and Y are the coodinates of the spot on the LEP and correspond to the error signals. (a) Movement induced on the solar beam by the zenithal stage. (b) Movement induced on the solar beam by the azimuthal stage.

Figure 6 :

 6 Figure6: Rotation during the day of the altazimuthal system in the coordinate system of the LEP. The servo control is temporally turned off and azimuthal stage is manually rotated to highlight the rotation of the projection in the LEP reference of the azimuthal axis. As the zenithal axis is always perpendicular to azimuthal one, we can deduce the rotation of the projection of the zenithal axis. X and Y correspond to the axis of the LEP. x and y are the axis of the altazimuthal system. x (respectively y) depends on X and Y. So it involves a coupled error signals generation.

Figure 7 :

 7 Figure 7: Determination of uncoupled error signals for azimuthal and zenithal stages servo control. (a) Calibration of ϕ depending of the azimuthal angle value. (b) Principle of conversion of (X,Y) coordinates into (x,y) coordinates. X and Y correspond to coupled error signals, whereas x and y are uncoupled error signals.

Figure 8 :

 8 Figure 8: (a) Servo-controlled suntracking based on the coordinate system rotation of the heliostat. X is the first cartesian coordinate respect to the center of the LEP. Its behavior is similar to a conventional PID-based servo control. (b) Stability of the solar spot at the top of the skylight (L 0 ≈ 1 m) and after focusing using a BaF 2 lens of 12.5 cm-focal length. (c) Beam stability down the skylight (L ≈ 10 m) and after focusing using another BaF 2 lens of 50 mm-focal length. Resolution of the LEP : 0.8 µm.

Figure 9 :

 9 Figure 9: (a) Servo-controlled suntracking based on the coordinate system rotation of the heliostat. Y is the second cartesian coordinate respect to the center of the LEP. As for the X coodinate, its behavior is similar to a conventional PID-based servo control. (b) Stability of the solar spot at the top of the skylight (L 0 ≈ 1 m) and after focusing using a BaF 2 lens of 12.5 cm-focal length. (c) Beam stability down the skylight (L ≈ 10 m) and after focusing using another BaF 2 lens of 50 mm-focal length. Resolution of the LEP : 0.8 µm.

Figure 10 :

 10 Figure 10: (Top) Experimental spectrum recorded at 14 m from the heliostat by a FTIR spectrometer (accumulation of 40 spectra for 10 min on a HgCdTe detector and with a resolution of 0.15 cm -1 ). (Middle) Non-convoluted simulated spectrum. (Bottom) Simulated spectrum convoluted by a 0.15 cm -1 -width Gaussian function. The simulations were performed with the 4A/OP software.

Figure 11 :

 11 Figure 11: Contribution of some atmospheric gases (N 2 O, CO 2 , H 2 O and O 3 ) to the experimental absorption spectrum (FTIR measurement by the accumulation of 40 spectra for 10 min on a HgCdTe detector and with a resolution of 0.15 cm -1 ). As on Figure 10, the simulations are computed with the 4A/OP software.

Acknowledgements

This work was funded by the ANR ASTRID Project #ANR 12-ASTR-0028 called QUIGARDE (2013-2016). The authors are also indebted to the Délégation Générale de l'Armement and the Centre National de la Recherche Scientifique for financial support of the PhD of Marie-Hélène Mammez.