Didier Fraisse
email: didier.fraisse@uca.fr

Alexis Bred
email: alexis.bred@uca.fr

Catherine Felgines
email: catherine.felgines@uca.fr

François Senejoux
email: francois.senejoux@uca.fr

Screening and Characterization of Antiglycoxidant Anthocyanins from Vaccinium myrtillus Fruit Using DPPH and Methylglyoxal Pre-Column HPLC Assays

Keywords: anthocyanins, delphinidin, petunidin, cyanidin glycosides, bilberry, pre-column HPLC method, antioxidant, glycation, BSA/D-ribose assay

Vaccinium myrtillus fruit (bilberry) is well known for its high richness in anthocyanins, which may be responsible for its preventive effects on several oxidative and carbonyl stress-related pathologies. However, limited data are available regarding the antioxidant and antiglycative contributions of its constituents. Spectrometric analyses were performed to evaluate anthocyanin content, radical scavenging and antiglycative properties of an anthocyanin-rich extract from bilberries. Additionally, original DPPH and methylglyoxal pre-column HPLC methods were instigated to allow straightforward identification of the main contributors to radical and carbonyl trapping effects. Finally, representative pure anthocyanins were evaluated using classical DPPH and antiglycation assays. Delphinidin, petunidin and cyanidin glycosides were identified as the most effective radical scavenging constituents in both HPLC and spectrometric DPPH evaluations. Potent antiglycative activities were also assessed for cyanidin, delphinidin and petunidin glucosides as attested by their respective IC 50 values of 114.2 ± 7.8, 130.5 ± 2.8, and 132.4 ± 3.7 µM. Interestingly, methylglyoxal spiking evaluation demonstrated that all bilberry anthocyanins exerted noticeable and comparable α-dicarbonyl trapping effects. Anthocyanins can be regarded as potent antiglycoxidant agents that might account for some health benefits of bilberries consumption. Besides, significant differences in their contributions were successfully highlighted by the employed pre-column HPLC assays.

Introduction

Vaccinium myrtillus L. (bilberry) is a low-branched shrub that belongs to the Ericaceae family. Also known as the European blueberry, this species produces dark blue edible fruits that can be eaten raw or cooked. A large variety of phenolic constituents has been described in V. myrtillus berries. Indeed, numerous flavonols including quercetin, myricetin, and isorhamnetin derivatives as well as tannins and phenolic acids have been reported [START_REF] Prokop | Effect of bilberry extract (Vaccinium myrtillus L.) on drug-metabolizing enzymes in rats[END_REF][START_REF] Rohloff | Nutritional composition of bilberries (Vaccinium myrtillus L.) from forest fields in Norway-Effects of geographic origin, climate, fertilization and soil properties[END_REF]. Additionally, these fruits are considered as one of the most important sources of anthocyanins, with contents ranging from 100 mg to 500 mg per 100 g of fresh weight [START_REF] Popović | Antioxidant and proapoptotic effects of anthocyanins from bilberry extract in rats exposed to hepatotoxic effects of carbon tetrachloride[END_REF]. Of interest, it has been demonstrated that such constituents exert a wide range of biological activities including potent radical scavenging and anti-inflammatory effects [START_REF] Szymanowska | Antioxidant and Potentially Anti-Inflammatory Activity of Anthocyanin Fractions from Pomace Obtained from Enzymatically Treated Raspberries[END_REF][START_REF] Miguel | Anthocyanins: Antioxidant and/or anti-inflammatory activities[END_REF]. More recently, several anthocyanin-rich extracts have been reported to inhibit in vitro formation of advanced glycation end products (AGEs) [START_REF] Harris | Investigating Wild Berries as a Dietary Approach to Reducing the Formation of Advanced Glycation Endproducts: Chemical Correlates of In Vitro Antiglycation Activity[END_REF][START_REF] Chen | Anthocyanins in blackcurrant effectively prevent the formation of advanced glycation end products by trapping methylglyoxal[END_REF]. Of interest, the antiglycative activity of bilberries was further suggested by a clinical study reporting that human plasma protein-bound AGEs can be partly reduced by V. myrtillus extract supplementation [START_REF] Chen | Effect of Vaccinium myrtillus Extract Supplement on Advanced Glycation End-products: A Pilot Study (P06-098-19)[END_REF]. All these reported activities might explain, at least in part, the positive effects of anthocyanin intake on human health. Indeed, several epidemiological or clinical data have highlighted the preventive effects of anthocyanin-rich food on oxidative stress and chronic pathologies such as cardiovascular diseases and diabetes [START_REF] Habanova | Intake of bilberries (Vaccinium myrtillus L.) reduced risk factors for cardiovascular disease by inducing favorable changes in lipoprotein profiles[END_REF][START_REF] Li | Purified Anthocyanin Supplementation Reduces Dyslipidemia, Enhances Antioxidant Capacity, and Prevents Insulin Resistance in Diabetic Patients[END_REF].

The antiglycative and antioxidant properties of anthocyanins are assumed to play an important role in their benefits on human health. However, the activities of such constituents can strongly differ depending on their structures [START_REF] Ali | Antiradical and reductant activities of anthocyanidins and anthocyanins, structure-activity relationship and synthesis[END_REF]. Hence, a basic estimation of total anthocyanin content from plants, fruits, or extracts might not efficiently reflect their biological effects. An important issue in the process of standardization of anthocyanin-containing products requires the characterization of most active constituents to provide adequate markers for quality control. Of interest, such a procedure is of particular importance concerning V. myrtillus in view of the numerous reported cases of adulteration of marketed products [START_REF] Gardana | Bilberry adulteration: Identification and chemical profiling of anthocyanins by different analytical methods[END_REF]. The conventional bio-guided fractionation of plant extracts is a long-lasting, labor-intensive, and costly task. Consequently, to hasten the identification of bioactive compounds, original and effective methods have been developed to allow straightforward detection of potential antioxidant and antiglycative constituents from complex plant matrices. For instance, HPLC analysis coupled with pre-column DPPH assay has been successfully applied in several phytochemical investigations [START_REF] Yao | Screening and quantitative analysis of antioxidants in the fruits of Livistona chinensis R. Br using HPLC-DAD-ESI/MS coupled with pre-column DPPH assay[END_REF]. This method consists of spiking extracts with a DPPH • solution prior to chromatographic evaluation and relies on the ability of antioxidant constituents to be transformed upon reaction with radical species. On this basis, the peak area of bioactive compounds is diminished in the chromatogram of spiked extracts, whereas inactive constituents are not affected by any modifications. This kind of procedure has been similarly applied to other radical species including ABTS •+ [START_REF] Shui | An improved method for the analysis of major antioxidants of Hibiscus esculentus Linn[END_REF], nitric oxide [START_REF] Fraisse | A Novel HPLC Method for Direct Detection of Nitric Oxide Scavengers from Complex Plant Matrices and Its Application to Aloysia triphylla Leaves[END_REF], and peroxynitrite scavengers [START_REF] Könczöl | Determination of Peroxynitrite Scavenging Activity of Phenols from Salvia spp[END_REF]. In the same way, HPLC spiking experiments have also been designed to seek out natural products capable of reacting with α-dicarbonyl compounds such as methylglyoxal (MGO) or glyoxal [START_REF] Tang | Pre-column incubation followed by fast liquid chromatography analysis for rapid screening of natural methylglyoxal scavengers directly from herbal medicines: Case study of Polygonum cuspidatum[END_REF]. Generated through different pathways including non-enzymatic degradation of reducing sugars, these highly reactive carbonyl species are indeed reported to be key intermediates in the formation of AGEs [START_REF] Nowotny | Advanced Glycation End Products and Oxidative Stress in Type 2 Diabetes Mellitus[END_REF].

The present study was thus aimed at performing a qualitative and quantitative chemical investigation of an anthocyanin-rich extract from V. myrtillus (AEVM) by HPLC analyses. Additionally, spectrophotometric and fluorimetric evaluations were achieved to estimate its total anthocyanin content and radical scavenging properties as well as its inhibition effects on the formation of AGEs. Finally, a different set of HPLC spiking experiments were performed in order to directly identify the main contributors to the antiglycoxidant (i.e., antioxidant and antiglycative) activities of the studied extract.

Materials and Methods

Plant Material and Reagents

Bilberry anthocyanin-rich dry extract, Anthocyan ® , was supplied by Ferlux SA (Cournon d'Auvergne, France). Methanol was of analytical grade and purchased from Carlo Erba Reagents SAS (Val-de-Reuil, France). Acetonitrile (MeCN) was of chromatographic grade (Carlo Erba Reagents SAS, Val-de-Reuil, France). All aqueous solutions were prepared with pure water produced by Milli-Q water (18.2 MΩ) system (Merck, Darmstadt, Germany). Phosphoric acid (purity 85%) was purchased from VWR Prolabo (Fontenay-sous-Bois, France). Delphinidin 3-O-glucoside chloride, cyanidin 3-O-glucoside chloride, cyanidin 3-O-galactoside chloride, petunidin 3-O-glucoside chloride, peonidin 3-O-glucoside chloride, malvidin 3-O-glucoside chloride, and malvidin 3-O-galactoside chloride were purchased from Extrasynthese (Genay, France). Aminoguanidine chloride, MGO (40% in water), bovine serum albumin (BSA), D-ribose, 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) and DPPH were bought from Sigma-Aldrich Chemical (Saint-Quentin Fallavier, France). MGO and DPPH radical solutions were freshly prepared every day and kept protected from light.

Spectrometric Evaluations of Total Anthocyanin Content, Radical Scavenging, and Antiglycation Activities

Total anthocyanin content was estimated using the colorimetric method following European Pharmacopoeia procedure [START_REF] Fresh | European Pharmacopoeia[END_REF]. The amount of total anthocyanins was expressed as milligram of cyanidin 3-O-glucoside equivalent per gram of dry extract.

DPPH radical scavenging activity was spectrophotometrically assessed according to a previously published method, with slight modifications [START_REF] Ndoye | Antioxidant and antiglycation properties of two mango (Mangifera indica L.) cultivars from Senegal. Asian Pac[END_REF]. Briefly, sample solutions (pure compounds or extract) were extemporaneously prepared at concentrations between 0.05 mg/mL and 1 mg/mL in water, and 20 µL of each were mixed to 2.5 mL of fresh DPPH solution (25 µg/mL in methanol). After 30 min of incubation at room temperature, absorbance was measured at 515 nm using a UV-vis Jasco V-630 spectrophotometer (Lisses, France). A standard curve of Trolox in the range of 0.1-3 mM was constructed (R 2 = 0.9978, y = 2752.8 x + 1.5196). Radical scavenging activities were respectively expressed in term of micromoles of Trolox equivalent (µmol TE) per gram of dry extract for AEVM and as IC 50 for pure compounds.

Inhibition of AGEs formation was determined using BSA/D-ribose assay as previously reported by Derbré et al. [START_REF] Séro | Tuning a 96-Well Microtiter Plate Fluorescence-Based Assay to Identify AGE Inhibitors in Crude Plant Extracts[END_REF]. Briefly, 40 µL of D-ribose (120 mM), 40 µL BSA (25 mg/mL), and 20 µL of the extract or pure compounds at different concentrations were incubated at 37 • C in a phosphate buffer, 50 mM, pH 7.4. After an incubation of 24 h, AGEs fluorescence was evaluated with a TECAN infinite F200 PRO microplate reader (Lyon, France) using 370 nm and 440 nm as the excitation and emission wavelengths. Aminoguanidine chloride was chosen as positive control, and results were indicated as IC 50 values in µg/mL for extract and in µM for pure compounds.

HPLC Analysis of AEVM

HPLC analysis of the studied extract was performed using a LaChrom Elite system (VWR-Hitachi, Radnor, PA, USA) equipped with two L7100 pumps, a L7200 autosampler, a L2450 diode array detector (DAD) performing the wavelength scanning from 200 nm to 600 nm, and EZ Chrom Elite software (Agilent Technologies, Massy, France). AEVM (1 mg/mL) was chromatographed with a reversed phase Interchim ® UP3ODB C 18 column (150 × 4.6 mm, 5 µm particle size). Gradient elution was developed with a mobile phase composed of water containing 1% phosphoric acid (A) and MeCN (B). The gradient was set as follows: 0-10 min, 9% B; 10-25 min, 9-12% B; 25-40 min, 12-16% B; 40-45 min, 16% B; 45-50 min, 16-40% B; 50-55 min, 9% B. A flow rate of 1 mL/min and an injection volume of 20 µL were selected. The quantification of individual anthocyanins was achieved by constructing calibration curves with commercial anthocyanin constituents that were representative of the five different groups occurring in bilberry (delphinidin, cyanidin, petunidin, malvidin and peonidin 3-O-glucosides). Standard solutions in the concentration range of 5-100 µg/mL were injected, and the chromatograms were monitored at a wavelength of 530 nm. Five-point calibration curves were constructed, and all of them exhibited satisfying linearities (R 2 > 0.99). Results were expressed as mg of constituent per g of dry extract.

Pre-column DPPH-HPLC Evaluation of AEVM

DPPH-HPLC experiments were conducted as previously described by Meda et al. [START_REF] Meda | Characterization of antioxidants from Detarium microcarpum Guill. et Perr. leaves using HPLC-DAD coupled with pre-column DPPH assay[END_REF] with minor modifications of extract and DPPH concentrations. Briefly, solution of AEVM (1.5 mg/mL) were spiked with DPPH solutions of different concentrations (0.75 mM and 1.5 mM in MeOH) at the ratio of 2:1 (v/v). The mixtures were then vortexed and incubated for 30 min at room temperature prior to HPLC analysis using the above-described conditions. The control consisted of a solution of AEVM (1.5 mg/mL) mixed with MeOH at a ratio of 2:1 (v/v) and then similarly treated than DPPH spiked solutions. The percentage of remaining constituents was calculated as the ratio between the constituent peak area after reaction with DPPH divided by the constituent peak area in the control sample.

Pre-column MGO-HPLC Evaluation of AEVM

MGO-HPLC experiments were performed according to a previously published method [START_REF] Tang | Pre-column incubation followed by fast liquid chromatography analysis for rapid screening of natural methylglyoxal scavengers directly from herbal medicines: Case study of Polygonum cuspidatum[END_REF] with slight modifications. Extemporaneously prepared solutions of AEVM (2 mg/mL in 50 mM phosphate buffer, pH = 7.4) were mingled with MGO solution (0.5 mM in phosphate buffer) at the ratio of 1:1 (v/v). The solutions were then vortexed and incubated for 1 h at 37 • C prior to HPLC analysis using the above reported method. The control contained a mixture of AEVM (2 mg/mL in phosphate buffer) and phosphate buffer at the ratio of 1:1 (v/v), which was prepared and incubated in the same way as the MGO spiked solutions. The percentage of remaining constituents was calculated as the ratio between the constituent peak area after reaction with MGO compared to the constituent peak area in control sample.

Statistical Analyses

The statistical significance of difference was analyzed by one-way ANOVA followed by Fisher's Least Significant Difference (LSD) test and p values of 0.05 or less (p ≤ 0.05) were considered statistically significant. All data are indicated as the mean ± standard error of the mean (SEM). The AGEs assays were performed in quadruplicate, and all other spectroscopic and chromatographic analyses were done in triplicate.

Results and Discussion

Total Anthocyanin Content, DPPH Scavenging, and Antiglycative Activities of AEVM

Considering that anthocyanin constituents are regarded as major contributors to the antioxidant activity and health benefits of bilberries [START_REF] Chen | Effect of Vaccinium myrtillus Extract Supplement on Advanced Glycation End-products: A Pilot Study (P06-098-19)[END_REF], an enriched extract was employed for the present investigation. Spectrometric estimation of its total anthocyanin content revealed a very high amount with an estimated value of 392.7 ± 7.7 mg of cyanidin 3-O-glucoside equivalents per gram of dry extract. The antioxidant properties of AEVM were further confirmed by performing a DPPH colorimetric assay. Indeed, the studied extract exerted very potent DPPH radical scavenging properties as ascertained by its activity of 3438 ± 129 µM of TE/g of dry extract. Such results are consistent with previous reports highlighting the powerful antioxidant effect of bilberry extracts using DPPH assay and other antioxidant models such as Oxygen Radical Absorbance Capacity (ORAC) or Ferric Reducing Antioxidant Power (FRAP) evaluations [START_REF]Functional Foods-Biochemical & Processing Aspects[END_REF][START_REF] Brasanac-Vukanovic | Wild Bilberry (Vaccinium myrtillus L., Ericaceae) from Montenegro as a Source of Antioxidants for Use in the Production of Nutraceuticals[END_REF]. By contrast, very limited data are available regarding antiglycative properties of V. myrtillus fruits. By using a BSA/D-ribose assay, our experiments demonstrated that AEVM induced a potent inhibitory effect on AGEs formation. With an IC 50 value of 71.69 ± 1.47 µg/mL, this extract exerted a significantly higher (p < 0.05) antiglycation effect than the positive control aminoguanidine chloride (264.7 ± 13.8 µg/mL). To the best of our knowledge, this is the first report of such activity for bilberries. This result can be tied in with previously published data on the potent antiglycation properties of fruit extracts from other Vaccinium species, such as V. cylindraceum, V. padifolium, and V. angustifolium [START_REF] Spínola | Hypoglycemic, anti-glycation and antioxidant in vitro properties of two Vaccinium species from Macaronesia: A relation to their phenolic composition[END_REF][START_REF] Mcintyre | Seasonal phytochemical variation of anti-glycation principles in lowbush blueberry (Vaccinium angustifolium)[END_REF]. Taken together, these preliminary results confirmed that AEVM exerted potent antioxidant and antiglycation properties, which might be attributable to its high anthocyanin content. HPLC experiments were thus implemented to determine the implication of each anthocyanin derivatives to the observed activities.

HPLC Characterization and Quantification of the Main Constituents from AEVM

Reversed-phase HPLC is a commonly employed technique for separation and analysis of complex mixtures of anthocyanins [START_REF] Da Costa | Analysis of anthocyanins in foods by liquid chromatography, liquid chromatography-mass spectrometry and capillary electrophoresis[END_REF]. It is worth mentioning that most published protocols report the use of highly acidic mobile phases. Indeed, such a marked acidification has been shown to allow more sensitive detection as well as better separation by generating the charged and colored flavylium form of anthocyanin derivatives [START_REF] Deineka | Regularities of Anthocyanins Retention in RP HPLC for "Water-Acetonitrile-Phosphoric Acid" Mobile Phases[END_REF]. Consequently, mixtures of phosphoric acid, water and acetonitrile have been widely investigated to highlight the constancies in the chromatographic behavior of this class of phenolics [START_REF] Deineka | Determination of Anthocyanins by High-Performance Liquid Chromatography: Regularities of Retention[END_REF]. Therefore, by using that kind of mobile phase composition and under the most favorable gradient of elution, a suitable resolution was obtained and all the major peaks of AEVM reached base-line separation (Figure 1).

Antioxidants 2020, 9, 512 5 of 11 and under the most favorable gradient of elution, a suitable resolution was obtained and all the major peaks of AEVM reached base-line separation (Figure 1). Analysis of UV-spectral data as well as comparison with anthocyanin standards and previous chemical investigations led to the characterization of 14 major anthocyanidin glycosides [START_REF] Mauray | Nutrigenomic analysis of the protective effects of bilberry anthocyanin-rich extract in apo E-deficient mice[END_REF][START_REF] Mauray | Bilberry anthocyanin-rich extract alters expression of genes related to atherosclerosis development in aorta of apo E-deficient mice[END_REF]. Consistently with previous reports on V. myrtillus composition [START_REF] Popović | Antioxidant and proapoptotic effects of anthocyanins from bilberry extract in rats exposed to hepatotoxic effects of carbon tetrachloride[END_REF], a substantial diversity of anthocyanidin structures was noticed as attested by the presence of delphinidin, cyanidin, petunidin, malvidin and peonidin glycosides (Table 1). Indeed, three different 3-O-glycosides of delphinidin were detected, including galactoside (1), glucoside (2), and arabinoside (4). Analogous glycosylation patterns and retention orders were also observed for cyanidin (3, 5, and 7) and petunidin (6, 8, and 10) constituents. In addition, peonidin 3-O-galactoside (9) as well as 3-O-glucoside and 3-Oarabinoside derivatives of malvidin (12 and 13) were identified. As previously reported for this same extract [START_REF] Mauray | Nutrigenomic analysis of the protective effects of bilberry anthocyanin-rich extract in apo E-deficient mice[END_REF][START_REF] Mauray | Bilberry anthocyanin-rich extract alters expression of genes related to atherosclerosis development in aorta of apo E-deficient mice[END_REF], a co-elution occurred for peonidin 3-O-glucoside [START_REF] Ali | Antiradical and reductant activities of anthocyanidins and anthocyanins, structure-activity relationship and synthesis[END_REF] and malvidin 3-O-galactoside (11′) explaining the lack of quantitative analysis for these two constituents. From a quantitative point of view, compounds 1 and 2 were shown to be the most abundant ones with respective contents of 45.18 mg/g and 45.07 mg/g of dry extract. Additionally, with global content of 125.36 mg/g, delphinidin glycosides were the most represented anthocyanin group among AEVM. Analysis of UV-spectral data as well as comparison with anthocyanin standards and previous chemical investigations led to the characterization of 14 major anthocyanidin glycosides [START_REF] Mauray | Nutrigenomic analysis of the protective effects of bilberry anthocyanin-rich extract in apo E-deficient mice[END_REF][START_REF] Mauray | Bilberry anthocyanin-rich extract alters expression of genes related to atherosclerosis development in aorta of apo E-deficient mice[END_REF]. Consistently with previous reports on V. myrtillus composition [START_REF] Popović | Antioxidant and proapoptotic effects of anthocyanins from bilberry extract in rats exposed to hepatotoxic effects of carbon tetrachloride[END_REF], a substantial diversity of anthocyanidin structures was noticed as attested by the presence of delphinidin, cyanidin, petunidin, malvidin and peonidin glycosides (Table 1). Indeed, three different 3-O-glycosides of delphinidin were detected, including galactoside (1), glucoside (2), and arabinoside (4). Analogous glycosylation patterns and retention orders were also observed for cyanidin (3, 5, and 7) and petunidin (6, 8, and 10) constituents. In addition, peonidin 3-O-galactoside (9) as well as 3-O-glucoside and 3-O-arabinoside derivatives of malvidin (12 and 13) were identified. As previously reported for this same extract [START_REF] Mauray | Nutrigenomic analysis of the protective effects of bilberry anthocyanin-rich extract in apo E-deficient mice[END_REF][START_REF] Mauray | Bilberry anthocyanin-rich extract alters expression of genes related to atherosclerosis development in aorta of apo E-deficient mice[END_REF], a co-elution occurred for peonidin 3-O-glucoside [START_REF] Ali | Antiradical and reductant activities of anthocyanidins and anthocyanins, structure-activity relationship and synthesis[END_REF] and malvidin 3-O-galactoside [START_REF] Ali | Antiradical and reductant activities of anthocyanidins and anthocyanins, structure-activity relationship and synthesis[END_REF] explaining the lack of quantitative analysis for these two constituents. From a quantitative point of view, compounds 1 and 2 were shown to be the most abundant ones with respective contents of 45.18 mg/g and 45.07 mg/g of dry extract. Additionally, with global content of 125.36 mg/g, delphinidin glycosides were the most represented anthocyanin group among AEVM.

DPPH Spiking Experiments for Detection of Main Radical Scavengers from AEVM

DPPH spiking assay has been described as a rapid and reliable method to detect antioxidant constituents in complex biological matrices [START_REF] Zhang | Comparative evaluation of three methods based on high-performance liquid chromatography analysis combined with a 2,2 -diphenyl-1-picrylhydrazyl assay for the rapid screening of antioxidants from Pueraria lobata flowers[END_REF]. This kind of experiment is based on the fact that radical scavenging compounds are structurally modified after reaction with radical species. Consequently, the peak area of bioactive constituents is lessened in the HPLC chromatogram of DPPH pretreated extracts. Spiking experiments with a 0.25 mM concentration of radical resulted in a significant decrease (p < 0.05) of peak areas of compounds 1, 2, 3, 4, 5, 6, 7, 8, and 10 (Figure 2). All of these constituents corresponded to delphinidin (1, 2, and 4), cyanidin (3, 5, and 7) or petunidin (6, 8, and 10) glycosides. By contrast, peonidin [START_REF] Habanova | Intake of bilberries (Vaccinium myrtillus L.) reduced risk factors for cardiovascular disease by inducing favorable changes in lipoprotein profiles[END_REF] and malvidin (12 and 13) derivatives were not impaired by radical pretreatment at that concentration. Of interest, data showed that no significant differences were observed in the reactivity of derivatives with analogous aglycones, indicating that the nature of the sugar moiety might not influenced the antioxidant activity of bilberry anthocyanins. Besides, a second experiment performed with a higher concentration of DPPH radical (0.5 mM) led to a significant modification to the peak areas of all the compounds of the extract. This implies that every anthocyanin constituent of AEVM can be considered as potential radical scavengers. Therefore, it appears that their effects noticeably differ depending on the substitution pattern of their aglycone moieties. Indeed, as illustrated in Figure 2, data indicated that delphinidin glycosides were the most affected compounds followed by petunidin and cyanidin derivatives. By contrast, minor diminutions were observed for malvidin and peonidin glycosides, suggesting that these two groups of anthocyanins might exert lower DPPH radical scavenging activities. From a structural point of view, it is interesting to note that the degree of hydroxylation in the B ring markedly affects the reactivity of these constituents. Indeed, with three adjacent hydroxyl groups in the 3 , 4 , and 5 positions of ring B (i.e., pyrogallol pattern), delphinidin derivatives were demonstrated to be the most reactive components of the extract. They were followed by petunidin and cyanidin glycosides, which are only hydroxylated in positions 3 and 4 . As reported above, these catechol-type anthocyanins were also deeply affected by radical pretreatment. Conversely, compounds bearing a single free hydroxyl group in position 4 (malvidin and peonidin glycosides) were shown to be the least reactive ones. Of interest, such observation is consistent with several previous investigations of structure-DPPH radical scavenging activity relationships investigations indicating that the radical scavenging activity of anthocyanin and flavonoid derivatives is increased with the number of hydroxyl groups on B ring [START_REF] Ali | Antiradical and reductant activities of anthocyanidins and anthocyanins, structure-activity relationship and synthesis[END_REF][START_REF] Amic | SAR and QSAR of the Antioxidant Activity of Flavonoids[END_REF][START_REF] Blando | Radical Scavenging and Anti-Inflammatory Activities of Representative Anthocyanin Groupings from Pigment-Rich Fruits and Vegetables[END_REF].

of anthocyanin and flavonoid derivatives is increased with the number of hydroxyl groups on B ring [START_REF] Ali | Antiradical and reductant activities of anthocyanidins and anthocyanins, structure-activity relationship and synthesis[END_REF][START_REF] Amic | SAR and QSAR of the Antioxidant Activity of Flavonoids[END_REF][START_REF] Blando | Radical Scavenging and Anti-Inflammatory Activities of Representative Anthocyanin Groupings from Pigment-Rich Fruits and Vegetables[END_REF].

MGO Spiking Experiments for Detection of Main Dicarbonyl Trapping Constituents from AEVM

α-dicarbonyl compounds such as MGO are considered to act as major contributors to AGE formation in foods as well as in vivo [START_REF] Chen | Effect of Vaccinium myrtillus Extract Supplement on Advanced Glycation End-products: A Pilot Study (P06-098-19)[END_REF][START_REF] Wang | Chemical Hazards in Thermally-Processed Foods[END_REF]. Interestingly, direct trapping of these highly reactive carbonyl species has been shown to constitute an effective strategy for limiting AGEs formation [START_REF] Chen | Anthocyanins in blackcurrant effectively prevent the formation of advanced glycation end products by trapping methylglyoxal[END_REF]. Therefore, the application of a method allowing straightforward detection of MGO scavenging constituents from complex plant extracts is highly valuable in order to characterize their active principles but also to clarify further the mechanism of their antiglycative action. As indicated in Figure 3, incubation of AEVM with 0.25 mM of MGO elicited a significant decrease in the areas of the 12 evaluated compounds (p < 0.05), confirming the antiglycation properties of AEVM constituents. However, unlike during DPPH spiking experiments, all the anthocyanins were similarly affected by MGO pretreatment, suggesting that they presented equivalent carbonyl trapping capacities. It also tends to reveal that structural differences among these derivatives might not impact their efficiencies. Such results are consistent with a previous investigation of Blackcurrant anthocyanins, which reported that 3-O-rutinosides of delphinidin and cyanidin both showed significant and comparable MGO scavenging capacities [START_REF] Chen | Anthocyanins in blackcurrant effectively prevent the formation of advanced glycation end products by trapping methylglyoxal[END_REF]. Of interest, by contrast with antiradical activities, the carbonyltrapping properties of flavonoid derivatives have been shown to not correlate with the presence of vicinal phenolic functions on B ring such as catechol or pyrogallol substitution patterns [START_REF] Shao | Essential Structural Requirements and Additive Effects for Flavonoids to Scavenge Methylglyoxal[END_REF]. Indeed, numerous investigations have determined that meta-dihydroxyl substituted aromatic nucleus constitutes the main site of scavenging action of flavonoids. Such patterns have been shown to generate nucleophilic site between to two phenolic groups that can be attacked by the electrophilic aldehyde group of MGO [START_REF] Hwang | Anti-glycation, Carbonyl Trapping and Anti-inflammatory Activities of Chrysin Derivatives[END_REF]. In line with this, structural analyses of MGO adducts of several common flavonoid derivatives including quercetin, apigenin and genistein have confirmed that positions 6 and 8 of their A ring were the major active sites for scavenging α-dicarbonyl compounds. It has to be noticed that all bilberry anthocyanins possess the same A-ring with 5,7-dihydroxyl substitution pattern, which likely constitutes the common structural feature of their carbonyl trapping properties.

MGO Spiking Experiments for Detection of Main Dicarbonyl Trapping Constituents from AEVM

α-dicarbonyl compounds such as MGO are considered to act as major contributors to AGE formation in foods as well as in vivo [START_REF] Chen | Effect of Vaccinium myrtillus Extract Supplement on Advanced Glycation End-products: A Pilot Study (P06-098-19)[END_REF][START_REF] Wang | Chemical Hazards in Thermally-Processed Foods[END_REF]. Interestingly, direct trapping of these highly reactive carbonyl species has been shown to constitute an effective strategy for limiting AGEs formation [START_REF] Chen | Anthocyanins in blackcurrant effectively prevent the formation of advanced glycation end products by trapping methylglyoxal[END_REF]. Therefore, the application of a method allowing straightforward detection of MGO scavenging constituents from complex plant extracts is highly valuable in order to characterize their active principles but also to clarify further the mechanism of their antiglycative action. As indicated in Figure 3, incubation of AEVM with 0.25 mM of MGO elicited a significant decrease in the areas of the 12 evaluated compounds (p < 0.05), confirming the antiglycation properties of AEVM constituents. However, unlike during DPPH spiking experiments, all the anthocyanins were similarly affected by MGO pretreatment, suggesting that they presented equivalent carbonyl trapping capacities. It also tends to reveal that structural differences among these derivatives might not impact their efficiencies. Such results are consistent with a previous investigation of Blackcurrant anthocyanins, which reported that 3-O-rutinosides of delphinidin and cyanidin both showed significant and comparable MGO scavenging capacities [START_REF] Chen | Anthocyanins in blackcurrant effectively prevent the formation of advanced glycation end products by trapping methylglyoxal[END_REF]. Of interest, by contrast with antiradical activities, the carbonyl-trapping properties of flavonoid derivatives have been shown to not correlate with the presence of vicinal phenolic functions on B ring such as catechol or pyrogallol substitution patterns [START_REF] Shao | Essential Structural Requirements and Additive Effects for Flavonoids to Scavenge Methylglyoxal[END_REF]. Indeed, numerous investigations have determined that meta-dihydroxyl substituted aromatic nucleus constitutes the main site of scavenging action of flavonoids. Such patterns have been shown to generate nucleophilic site between to two phenolic groups that can be attacked by the electrophilic aldehyde group of MGO [START_REF] Hwang | Anti-glycation, Carbonyl Trapping and Anti-inflammatory Activities of Chrysin Derivatives[END_REF]. In line with this, structural analyses of MGO adducts of several common flavonoid derivatives including quercetin, apigenin and genistein have confirmed that positions 6 and 8 of their A ring were the major active sites for scavenging α-dicarbonyl compounds. It has to be noticed that all bilberry anthocyanins possess the same A-ring with 5,7-dihydroxyl substitution pattern, which likely constitutes the common structural feature of their carbonyl trapping properties.

DPPH Radical Scavenging and Antiglycative Activities of Representative Anthocyanin Standards

To further confirm the interpretations of the performed spiking assays, 3-O-glucosides of delphinidin, cyanidin, petunidin, malvidin and peonidin were assessed for their antioxidant and antiglycative capacities. As indicated in Table 2, DPPH scavenging activities of these constituents were highly consistent with HPLC experiments. Indeed, with respective IC50 values of 10.55 ± 0.06 μM and 10.97 ± 0.12 μM, delphinidin and petunidin glucosides were confirmed to exert the most potent DPPH scavenging effects. Of interest, excellent activity was also highlighted for cyanidin glycoside as attested by its IC50 of 11.70 ± 0.08 μM. In the same way as for DPPH spiking experiments, weaker effects were observed for malvidin (IC50 = 13.33 ± 0.16 μM) and peonidin (IC50 = 14.87 ± 0.28 μM) derivatives. In addition, potent antiglycation activities were also pointed out for these five anthocyanidin glycosides, as attested by their IC50 values being substantially lower than that of aminoguanidine chloride. It is important to note that significant differences were observed among the evaluated pure compounds. Indeed, with respective IC50 values of 114.2 ± 7.8, 130.5 ± 2.8, and 132.4 ± 3.7 μM, cyanidin, delphinidin and petunidin glucosides induced the highest inhibitory effects on AGE formation. Besides, malvidin and peonidin derivatives were shown to exert slightly weaker inhibitory effects on AGE formation. Interestingly, these results are consistent with a previous structure-activity relationship evaluation of flavonoid constituents showing that methylation generally exert a negative influence on their antiglycation activity [START_REF] Xie | Structures Required of Polyphenols for Inhibiting Advanced Glycation end Products Formation[END_REF]. Considering their above-described analogous carbonyl trapping

DPPH Radical Scavenging and Antiglycative Activities of Representative Anthocyanin Standards

To further confirm the interpretations of the performed spiking assays, 3-O-glucosides of delphinidin, cyanidin, petunidin, malvidin and peonidin were assessed for their antioxidant and antiglycative capacities. As indicated in Table 2, DPPH scavenging activities of these constituents were highly consistent with HPLC experiments. Indeed, with respective IC 50 values of 10.55 ± 0.06 µM and 10.97 ± 0.12 µM, delphinidin and petunidin glucosides were confirmed to exert the most potent DPPH scavenging effects. Of interest, excellent activity was also highlighted for cyanidin glycoside as attested by its IC 50 of 11.70 ± 0.08 µM. In the same way as for DPPH spiking experiments, weaker effects were observed for malvidin (IC 50 = 13.33 ± 0.16 µM) and peonidin (IC 50 = 14.87 ± 0.28 µM) derivatives. In addition, potent antiglycation activities were also pointed out for these five anthocyanidin glycosides, as attested by their IC 50 values being substantially lower than that of aminoguanidine chloride. It is important to note that significant differences were observed among the evaluated pure compounds. Indeed, with respective IC 50 values of 114.2 ± 7.8, 130.5 ± 2.8, and 132.4 ± 3.7 µM, cyanidin, delphinidin and petunidin glucosides induced the highest inhibitory effects on AGE formation. Besides, malvidin and peonidin derivatives were shown to exert slightly weaker inhibitory effects on AGE formation. Interestingly, these results are consistent with a previous structure-activity relationship evaluation of flavonoid constituents showing that methylation generally exert a negative influence on their antiglycation activity [START_REF] Xie | Structures Required of Polyphenols for Inhibiting Advanced Glycation end Products Formation[END_REF]. Considering their above-described analogous carbonyl trapping capacities, these discrepancies suggests the existence of additional contributing mechanisms to the antiglycative action. Of interest, inhibitory effects on AGE formation positively correlated with DPPH radical scavenging activities (R 2 = 0.7695), indicating that the antioxidant capacity of bilberry anthocyanins might also be partly responsible for their antiglycative properties. It is worth mentioning that this dual mode of action has been previously reported for several phenolic derivatives, including gallocatechin derivatives and stilbene glucosides [START_REF] Ho | Dietary Phenolics as Reactive Carbonyl Scavengers: Potential Impact on Human Health and Mechanism of Action[END_REF][START_REF] Liu | Inhibition of Resveratrol Glucosides against AGEs Formation[END_REF].

Conclusions

The present study points out the efficiency of the two employed pre-column HPLC evaluations for direct detection of radical and carbonyl scavenging constituents from complex plant matrices. Besides, a DPPH spiking assay has successfully highlighted the importance of aglycone moieties in the individual antioxidant activities of bilberry anthocyanins. DPPH colorimetric evaluation of 3-O-glucosides of delphinidin, cyanidin, petunidin, malvidin, and peonidin further supports this observation. It indeed confirms that delphinidin, petunidin, and cyanidin derivatives exert the highest radical scavenging activities as attested by their respective IC 50 values of 10.55 ± 0.06, 10.97 ± 0.12, and 11.70 ± 0.08 µM. In addition, the MGO-HPLC assay has provided novel information regarding the mechanism of bilberry anthocyanins inhibitory action on AGEs formation and their α-dicarbonyl scavenging capacities. Taken together, these results indicate that anthocyanin constituents, and more especially delphinidin, petunidin, and cyanidin glycosides, can be regarded as potent contributors to the antiglycative properties of V. myrtillus fruits owing to a dual mode of action associating radical as well as carbonyl trapping effects. Hence, this investigation tends to support the potential benefits of anthocyanin consumption on both oxidative and carbonyl stresses. However, further in vivo investigations will be required to confirm the physiological relevance of these reported activities.

Figure 1 .

 1 Figure 1. HPLC profile of anthocyanin-rich extract from Vaccinium myrtillus fruit with detection at 530 nm. 1: delphinidin 3-O-galactoside, 2: delphinidin 3-O-glucoside, 3: cyanidin 3-O-galactoside, 4: delphinidin 3-O-arabinoside, 5: cyanidin 3-O-glucoside, 6: petunidin 3-O-galactoside, 7: cyanidin 3-O-arabinoside, 8: petunidin 3-O-glucoside, 9: peonidin 3-O-galactoside, 10: petunidin 3-Oarabinoside, 11: peonidin 3-O-glucoside, 11′: malvidin 3-O-galactoside, 12: malvidin 3-O-glucoside, 13: malvidin 3-O-arabinoside.

Figure 1 .

 1 Figure 1. HPLC profile of anthocyanin-rich extract from Vaccinium myrtillus fruit with detection at 530 nm. 1: delphinidin 3-O-galactoside, 2: delphinidin 3-O-glucoside, 3: cyanidin 3-O-galactoside, 4: delphinidin 3-O-arabinoside, 5: cyanidin 3-O-glucoside, 6: petunidin 3-O-galactoside, 7: cyanidin 3-O-arabinoside, 8: petunidin 3-O-glucoside, 9: peonidin 3-O-galactoside, 10: petunidin 3-O-arabinoside, 11: peonidin 3-O-glucoside, 11 : malvidin 3-O-galactoside, 12: malvidin 3-O-glucoside, 13: malvidin 3-O-arabinoside.

Figure 2 .

 2 Figure 2. (a) Percentage of remaining compounds after incubation of the extract with various concentrations of DPPH (0, 0.25, and 0.5 mM). Data are presented as means ± SEM (n = 3). * p < 0.05 vs. control (DPPH = 0 mM); (b) Heat map representation of the percentage of remaining compounds clustered according to their aglycone moieties: delphinidin (1, 2, 4), petunidin (6, 8, 10), cyanidin (3, 5, 7), peonidin (9), and malvidin (12, 13).

Figure 2 .

 2 Figure 2. (a) Percentage of remaining compounds after incubation of the extract with various concentrations of DPPH (0, 0.25, and 0.5 mM). Data are presented as means ± SEM (n = 3). * p < 0.05 vs. control (DPPH = 0 mM); (b) Heat map representation of the percentage of remaining compounds clustered according to their aglycone moieties: delphinidin (1, 2, 4), petunidin (6, 8, 10), cyanidin (3, 5, 7), peonidin (9), and malvidin (12, 13).

Figure 3 .

 3 Figure 3. Percentage of remaining compounds after incubation of the extract with methylglyoxal (MGO, 0.25 mM). Data are presented as means ± SEM (n = 3). *p < 0.05 vs. control (MGO = 0 mM).

Figure 3 .

 3 Figure 3. Percentage of remaining compounds after incubation of the extract with methylglyoxal (MGO, 0.25 mM). Data are presented as means ± SEM (n = 3). * p < 0.05 vs. control (MGO = 0 mM).

Table 1 .

 1 Retention time and UV maximum absorption of constituents of anthocyanin-rich extract from Vaccinium myrtillus fruit.

	Peak Number	Compound	Retention Time (min)	UV, λ max (nm)	Content (mg/g)
	1	Delphinidin 3-O-galactoside	7.6	277-524	45.18 ± 0.20
	2	Delphinidin 3-O-glucoside	9.4	277-524	45.07 ± 0.31
	3	Cyanidin 3-O-galactoside	11.7	280-517	29.69 ± 0.07
	4	Delphinidin 3-O-arabinoside	12.7	278-524	35.11 ± 0.37
	5	Cyanidin 3-O-glucoside	15.0	280-517	31.01 ± 0.08
	6	Petunidin 3-O-galactoside	16.1	278-524	21.00 ± 0.09
	7	Cyanidin 3-O-arabinoside	18.4	282-517	21.68 ± 0.51
	8	Petunidin 3-O-glucoside	19.5	277-524	35.08 ± 0.85
	9	Peonidin 3-O-galactoside	21.6	280-518	5.33 ± 0.10
	10	Petunidin 3-O-arabinoside	23.0	277-525	14.92 ± 0.09
	11 11	Peonidin 3-O-glucoside Malvidin 3-O-galactoside	25.4	277-522	N.D. N.D.
	12	Malvidin 3-O-glucoside	28.8	277-527	41.45 ± 0.68
	13	Malvidin 3-O-arabinoside	31.8	277-527	9.51 ± 0.25

N.D., not determined; content values are presented as means ± SEM (n = 3).

Table 2 .

 2 DPPH radical scavenging and antiglycation activities of representative anthocyanins from Vaccinium myrtillus fruit.

	Compound	DPPH radical scavenging activity (IC50, μM)	Antiglycation activity (IC50, μM)
	Delphinidin 3-O-glucoside	10.55 ± 0.06 a	130.5 ± 2.8 a
	Petunidin 3-O-glucoside	10.97 ± 0.12 a	132.4 ± 3.7 a,b
	Cyanidin 3-O-glucoside	11.70 ± 0.08 b	114.2 ± 7.8 a
	Malvidin 3-O-glucoside	13.33 ± 0.16 c	151.5 ± 6.0 b
	Peonidin 3-O-glucoside	14.87 ± 0.28 d	193.7 ± 11.7 c
	Trolox	17.62 ± 0.35 e	N.D.
	Aminoguanidine chloride	N.D.	2387.4 ± 123.7 d
	Data are presented as means ± SEM (n = 3-4). Values in the same column sharing identical superscript
	are not significantly different from each other (p > 0.05). N.D., not determined.

Table 2 .

 2 DPPH radical scavenging and antiglycation activities of representative anthocyanins from Vaccinium myrtillus fruit.

	Compound	DPPH Radical Scavenging Activity (IC 50 , µM)	Antiglycation Activity (IC 50 , µM)
	Delphinidin 3-O-glucoside	10.55 ± 0.06 a	130.5 ± 2.8 a
	Petunidin 3-O-glucoside	10.97 ± 0.12 a	132.4 ± 3.7 a,b
	Cyanidin 3-O-glucoside	11.70 ± 0.08 b	114.2 ± 7.8 a
	Malvidin 3-O-glucoside	13.33 ± 0.16 c	151.5 ± 6.0 b
	Peonidin 3-O-glucoside	14.87 ± 0.28 d	193.7 ± 11.7 c
	Trolox	17.62 ± 0.35 e	N.D.
	Aminoguanidine chloride	N.D.	2387.4 ± 123.7

d

Data are presented as means ± SEM (n = 3-4). Values in the same column sharing identical superscript are not significantly different from each other (p > 0.05). N.D., not determined.

Antioxidants 2020, 9, 512; doi:10.3390/antiox9060512 www.mdpi.com/journal/antioxidants

Funding: This research received no external funding.

Acknowledgments:

The authors would like to thank David Daroux (Ferlux SA) for plant extract supplying.

Conflicts of Interest:

The authors declare no conflict of interest.


Edith Ribot-Ciscar
email: edith.ribot-ciscar@univ-amu.fr

Rochelle Ackerley

Muscle proprioceptive feedback can be adapted to the behavioral and emotional context in humans

Keywords: muscle afferent, gamma drive, microneurography, humans

To interact with the external world, incoming sensory information informs us about situational changes that are of relevance to our behavior and we act upon these accordingly, be it in a produced movement or an internal reaction (e.g. an emotion, paying attention). Humans efficiently interact with their world and each other and to do this, a system is required to promote relevant sensory feedback to allow fast, accurate, and appropriate actions. We review findings from single -fusimotor system on their activity. This includes changes in firing from cognitive and emotional influences, which we postulate can prepare the body for responsive and appropriate action to a change in environment.

. The existence of this efferent system to control muscle spindle sensitivity leads us to believe that its function is useful, such as proprioceptive adaptation during a movement in progress.

Researchers in the field of proprioception have investigated which conditions engage the fusimotor system. The technique of microneurography (Box 2), which permits direct recordings from human peripheral nerves and access to the messages emitted by single afferents, has provided many insights into proprioceptive feedback [START_REF] Vallbo | Impulses recorded with micro-electrodes in human muscle nerves during stimulation of mechanoreceptors and voluntary contractions[END_REF][START_REF] Vallbo | Microneurography: how the technique developed and its role in the investigation of the sympathetic nervous system[END_REF]. Researchers have asked whether the fusimotor system allows a selective control of muscle spindle afferent sensitivity, which would allow us to adjust muscle proprioceptive feedback to the environmental context and the requirements of a motor activity [START_REF] Vallbo | Somatosensory, proprioceptive, and sympathetic activity in human peripheral nerves[END_REF].

In animals, it has been demonstrated that sensitivity of muscle afferents can be modified by descending influences of the -fusimotor drive, where the central nervous system can 'set' the activity level independently [START_REF] Prochazka | Fusimotor set": new evidence for alpha-independent control of gamma-motoneurones during movement in the awake cat[END_REF]. Similar mechanisms have been investigated in humans, but only small effects of a descending drive have been found [START_REF] Kakuda | Fusimotor and skeletomotor activities are increased with precision finger movement in man[END_REF][START_REF] Kakuda | Dynamic response of human muscle spindle afferents to stretch during voluntary contraction[END_REF][START_REF] Vallbo | Independence of skeletomotor and fusimotor activity in man?[END_REF]. Most microneurographic recordings showed a coupling between muscle spindle activity and muscle contraction (- coactivation), which led to the conclusion that the fusimotor system functioned to compensate for the slackening of receptors during muscle shortening [START_REF] Prochazka | Proprioceptive feedback and movement regulation[END_REF][START_REF] Vallbo | Human muscle spindle response in a motor learning task[END_REF]. Although fully in agreement with this view of the role of the fusimotor system, microneurography work from our group, performed at the level of the leg, has provided evidence that this role is not exclusive, but that independent control of the -fusimotor system can also exist, which has opened the door to other functions of the muscle proprioceptive system [START_REF] Ackerley | Seeing your foot move changes muscle proprioceptive feedback[END_REF][13][14][15][16]. We have found that it is imperative to control the environmental situation well and monitor the physiological state of the participant to ensure that these are stable, as during cognitive and emotional manipulation, a steady baseline is required. It is important for the participant to be comfortable in the environment and physiological signals of the participant (e.g. heart rate, electrodermal activity, electromyographic signals, see [13]) can be measured. These are useful during the whole microneurography processes, as fluctuations and high activity in these can indicate that the participant is not calm or comfortable, without having to continually explicitly ask them.

1-Evidence for an independent gamma drive in subjects at rest

a. Recordings from single -fusimotor neurons Our first data on the fusimotor system were obtained by the direct recording of -fusimotor neurons in subjects at rest [16], where we showed that these efferents were activated by factors such as cognitive, behavioral, and environmental interventions. This was in contrast to the skeletomotor extrafusal muscle efferents, which could be clearly distinguished during movements and were not influenced by such top-down factors. We also observed that when -fusimotor activity was triggered, the subject could voluntarily stop it by seeking a deep state of physical and mental relaxation. This showed the importance of controlling the relaxation of the subject, since if the gamma system was already activated because the subject was alert (e.g. interested in the experimental approach), any test maneuver would not increase the -fusimotor activity relative to the control situation in a clear or true manner (see [START_REF] Proske | The Proprioceptive Senses: Their Roles in Signaling Body Shape, Body Position and Movement, and Muscle Force[END_REF][START_REF] Zehr | What functions do reflexes serve during human locomotion?[END_REF] for further information about experimental controls), although data modeling can also aid in accounting for descending influences under active conditions [START_REF] Dimitriou | Human muscle spindles act as forward sensory models[END_REF][START_REF] Dimitriou | Enhanced muscle afferent signals during motor learning in humans[END_REF]. However, these recordings were rare and it took 5 years to collect data from the 6 -fibers presented in the paper. Communication with other microneurographers at the time revealed they had occasionally also encountered similar neurons. Characteristically, these neurons did not respond to sensory stimulation, and demonstrated intermittent behavioral changes with no clear cause. Due to the challenges in finding such activity and the difficulty in testing the neuron, it was not feasible to pursue our questioning of the influence of the -fusimotor system using direct -efferent recordings. Rather, as muscle spindle afferents are readily recorded, we sought to infer -activity through their influence on muscle spindles.

b. Recordings from single muscle spindle afferents during cognitive

processes By taking particular care of the mental state of the participants, we subsequently showed an increase in the responsiveness of muscle spindle primary endings to movement in resting subjects performing mental computation [START_REF] Ribot-Ciscar | Increased muscle spindle sensitivity to movement during reinforcement manoeuvres in relaxed human subjects[END_REF]. It was unlikely that these changes could be attributed to changes in muscle sympathetic activity, despite a direct sympathetic innervation of intrafusal muscle fibers [START_REF] Radovanovic | Sympathetic innervation of human muscle spindles[END_REF], since muscle spindle firing is not influenced by a strong and sustained physiological activation of muscle sympathetic outflow [START_REF] Macefield | Resting discharge of human muscle spindles is not modulated by increases in sympathetic drive[END_REF]. Rather, we postulated that switching from a relaxed state to an active mental state triggers a -fusimotor drive independent of the -motoneuron drive. Beyond this general excitatory effect, the question remained as to whether there could be a modulation of the static and/or Box 1. Muscle spindles: These are mechanoreceptors that respond to changes in the stretch of a muscle. Primary type Ia sensory fibers are the most numerous (signaling the degree of change in muscle movement), there are less secondary type II fibers (signaling the length of the muscle), and Golgi tendon organs (type Ib, sensing muscle tension) are even rarer. These messages are processed centrally and add to our sense of proprioception (kinesthesia), providing us with a sense of body position and self-movement.

Box 2. Microneurography:

This technique of percutaneously accessing peripheral nerve fibers in awake humans. In single unit microneurography, a needle electrode is inserted through the skin and into a peripheral nerve, where recordings from individual fibers, typically afferents, can be distinguished. Stimuli can be applied to the body, while single unit activity is monitored to different interventions. dynamic parameters of muscle proprioceptive feedback in the regulation of movements.

To answer this question, we investigated whether muscle proprioceptive sensitivity changed in a situation where the subject was asked to close their eyes and focus their attention on an imposed movement, in order to recognize it, as compared to a control situation where they did not pay attention to the movement [15]. We imposed movements in the shape of cursive letters because they are sufficiently complex to engage the subject's attention, but were easy to name once recognized. The neural response of muscle spindles to the same movement describing a letter was highly reproducible in the control condition; however, the afferent responses were modified when the subject paid attention to the movement to recognize it (Fig. 2A). The observed changes suggested that primary muscle spindle endings behaved like secondary endings. We interpreted this 'secondarization' as a means to facilitate the coding of the shape of the movement that would facilitate the task of recognizing letters. In all, the percentage of correct recognized movements increased when changes in muscle spindle sensitivity were observed. Thus, the -fusimotor drive seemed to adapt to select the most relevant muscle proprioceptive information according to the task.

It was postulated that such spindle sensitization could also be the result of an aspecific effect, such as arousal, which could condition the receptivity of the whole organism to surrounding stimuli. We therefore extended this research and asked the subject to pay attention either to the speed of ramp movements of their foot or to its final position reached [14]. We observed changes in muscle afferent firing when participants attended to the velocity or amplitude, reflecting dynamic and static gamma activation, respectively. These results support the view that -fusimotor control is not an aspecific effect but results from a task-specific event and allows the parametric control of muscle spindle feedback to fit task requirements. c. Recordings from single muscle spindle afferents during emotional processes It is not only cognitive processes that can engage the -fusimotor drive, but emotions can influence the feedback gained from muscle afferents. Emotions can evoke strong reactions that profoundly influence our bodies and modulate our preparedness to move [START_REF] Van Loon | Emotional stimuli modulate readiness for action: A transcranial magnetic stimulation study[END_REF], where pleasantness typically facilitates approach and unpleasantness primes withdrawal. The emotion derived from a situation can impact on the timing of movements [START_REF] Esteves | Motor planning of goaldirected action is tuned by the emotional valence of the stimulus: a kinematic study[END_REF], where unpleasant situations are particularly salient [START_REF] Coombes | Emotion and movement: Activation of defensive circuitry alters the magnitude of a sustained muscle contraction[END_REF][START_REF] Hillman | Emotion and motivated behavior: Postural adjustments to affective picture viewing[END_REF]. Recently we recorded muscle afferent activity [13], while changing the participant's emotional state through listening to happy, sad, or neutral-emotional music. Muscle afferent firing in response to passive ankle movements was modified by the emotional context, especially for the induced sad emotion, where the muscle spindle dynamic response increased (Fig. 2B). This effect could be seen in the change in the depth of modulation of muscle afferent firing over each condition (i.e. the maximum minus the minimum firing rate), where in the sad condition, there were typically marked pauses between the sinusoidal movements that were not present in the neutral condition. This was supported by behavioral findings showing that under the same conditions, kinesthetic acuity was also affected by emotional state, where it was improved during imposed sadness [START_REF] Samain-Aupic | Emotions can alter kinesthetic acuity[END_REF]. Our findings are also congruent with those obtained using transcranial magnetic stimulation, showing that corticospinal excitability is increased when listening to emotional music [START_REF] Giovannelli | The effect of music on corticospinal excitability is related to the perceived emotion: A transcranial magnetic stimulation study[END_REF]. The specific effect of emotions that we demonstrated may be exploited therapeutically, such as for priming movements in patients with depression, by listening to sad music and it is clear that emotions should be taken into account during movement investigations, especially as emotion is already known to shape our perception in vision and audition [START_REF] Brosch | The impact of emotion on perception, attention, memory, and decision-making[END_REF].

d. Recordings from single muscle spindle afferents during multisensory processes

The effect of the -fusimotor system can be seen when unisensory, as compared to multisensory, information is received. For example, we found that when participants were not able to see their foot move, muscle afference was slightly increased, as compared to when participants had congruent proprioceptive and visual information (Fig. 2C) [START_REF] Ackerley | Seeing your foot move changes muscle proprioceptive feedback[END_REF]. Similarly, when proprioceptive information from a moving hand was coupled with incongruent visual information, proprioceptive sensitivity was reduced to resolve bi-sensory conflict [START_REF] Jones | Proprioceptive feedback is reduced during adaptation to a visuomotor transformation: preliminary findings[END_REF]. Along the same lines, we would predict that in a rubber-hand illusion paradigm [START_REF] Tsakiris | The rubber hand illusion revisited: Visuotactile integration and self-attribution[END_REF], the embodiment of the fake hand is accompanied by a decrease in muscle afferent inputs due to a decrease in the -fusimotor drive, which counterpart would be to give more weight to the visual information of touching the plastic hand, but this remains to be explored. The specific contribution of muscle proprioceptive signals to body ownership has been recently demonstrated where grasping an artificial finger induced a sense of ownership of it [START_REF] Héroux | Time, touch and temperature affect perceived finger position and ownership in the grasp illusion[END_REF], as well as the influence of cognitive and environmental factors on brain process from these sensory inputs [START_REF] Qureshi | Effects of horizontal distance and limb crossing on perceived hand spacing and ownership: Differential sensory processing across hand configurations[END_REF].

2-Independent gamma motor control for purposeful movements adapted to

the situation The work summarized above and visualized in Figure 2 demonstrates the existence of an independent, top-down control of the -fusimotor system, since all changes have been shown in the resting subject, without concurrent -activity. Note that stronger modulation may have been predicted from animal work, but it has always been an intriguing observation that muscle spindle firing rates are much lower in humans than in animals [START_REF] Prochazka | Muscle afferent function and its significance for motor control mechanisms during voluntary movements in cat, monkey, and man[END_REF]. For example, the fusimotor-induced increase in human spindle discharge during isometric contraction may be ~15 times lower in humans than in awake cats [START_REF] Prochazka | Ia afferent activity during a variety of voluntary movements in the cat[END_REF][START_REF] Wilson | Discharge of human muscle spindle afferents innervating ankle dorsiflexors during target isometric contractions[END_REF]. Such differences may therefore be present in the influence of the fusimotor system on muscle spindle sensitivity and small changes in muscle spindle sensitivity may nevertheless have a significant effect on proprioception. This was the case during listening to sad music [13,[START_REF] Samain-Aupic | Emotions can alter kinesthetic acuity[END_REF] and during noise-induced increases in the variability of the spindle discharge [START_REF] Ribot-Ciscar | Noise-enhanced kinaesthesia: a psychophysical and microneurographic study[END_REF], both showing improved movement sense.

The selective and differential -fusimotor system that controls muscle spindle sensitivity is likely activated to adapt muscle proprioceptive feedback during voluntary motor activities. Although it is not easy to infer -activity from the recording of spindle endings in the active subject, a number of observations provide evidence of this. The finding that humans can finely-control muscle proprioceptive feedback through an optimized fusimotor drive is in line with the idea that proprioceptive training can improve motor performance [START_REF] Ostry | Sensory Plasticity in Human Motor Learning[END_REF]. There are also many studies showing that somatosensory factors play a major role in the process of learning, particularly at the early stage of motor skill acquisition [START_REF] Wong | Can proprioceptive training improve motor learning?[END_REF][START_REF] Bernardi | Somatosensory contribution to the initial stages of human motor learning[END_REF][START_REF] Vahdat | Structure of plasticity in human sensory and motor networks due to perceptual learning[END_REF]. In a direct way, Dimitriou showed an increase in the firing of muscle spindle afferents during a visuomotor task, reflecting a -fusimotor control that adjusted the muscle proprioceptive system in motor learning [START_REF] Dimitriou | Enhanced muscle afferent signals during motor learning in humans[END_REF]. In a comparable visuomotor rotation task, other authors found a decrease in spindle firing [START_REF] Jones | Proprioceptive feedback is reduced during adaptation to a visuomotor transformation: preliminary findings[END_REF], probably because of differences in experimental procedures, but both studies strongly suggest the presence of independent fusimotor control. Further, during simple block grasping with the thumb and finger, muscle afferent activity has been shown to better relate to the future muscle state, rather than the current state, demonstrating that an uncoupling of fusimotor and skeletomotor control would enable muscle spindles to work as a forward sensory model to predict the future kinematic activity of its muscle [START_REF] Dimitriou | Human muscle spindles act as forward sensory models[END_REF]. It is probable that focused movement training can increase proprioceptive acuity, which has been shown to be predictive of sport performance in elite athletes, such as dancers [START_REF] Han | Level of competitive success achieved by elite athletes and multi-joint proprioceptive ability[END_REF]. Our capacity to tune muscle proprioceptive feedback by optimizing gamma drive could also be a means to restore altered motor function [for a review, see 44].

In conclusion, it is clear that muscle proprioceptive feedback can be modulated by the -fusimotor drive, to adapt behavior to the current situation. Although few studies have been performed in this area, especially due to the difficulty in accessing muscle afferents under controlled environmental conditions, the evidence shows that both cognitive and emotional factors, as well as input from other senses, play a role in modifying the descending drive to influence muscle receptor sensitivity. This feedforward drive therefore allows the rapid and efficient adaptation to a change in situation that can help prepare the body for responsive and appropriate action. This study examined the change in sensitivity of muscle afferent firing during different emotional settings. The results showed that emotions can influence proprioceptive feedback, particularly when sadness was imposed, thus demonstrating the profound top-down influence that emotions can have on the body.

[14] E. Ribot-Ciscar, V. Hospod, J.P. Roll, J. Aimonetti, Fusimotor drive may adjust muscle spindle feedback to task requirements in humans., J. The study showed that muscle afferent activity is altered when attention is modulated, -fusimotor system. The majority of primary Ia muscle afferents changed when the participant was asked to focus on a pattern-recognition task of an imposed movement, showing that the body can up-regulate sensory muscle signals to aid in environmental demands. *[16] E. Ribot, J.P. Roll, J.P. Vedel, Efferent discharges recorded from single skeletomotor and fusimotor fibres in man., J. Physiol. 375 (1986) 251-68.

Figure 1 .

 1 Figure 1. Schematic of how the -fusimotor drive influences the responses of muscle spindles. The descending influence of process such as cognition, emotions, and other senses (shown in red) can directly influence the static and/or dynamic sensitivity of muscle spindles via the selective control of static and/or dynamic -fusimotor efferents. The effect of this can be measured by imposing the exact same movement to a muscle under different conditions, as measured via single unit microneurography recordings from muscle afferents.

Figure 2 .

 2 Figure 2. Examples of situations where there is a descending influence from the -fusimotor drive, as shown by changes in muscle afferent activity to imposed movements of the foot. (A) The imposition of a letter as a 2D movement without the participant paying attention (top) and when the participant was i nstructed to pay attention to the movement to attempt to recognize the letter (bottom). The muscle afferent response was more variable during recognition. Adapted from Hospod et al (2007) [15]. (B) Sinusoidal movements of the foot were imposed during listening to sad, neutral or happy music, to induce the corresponding emotion. During sad music, the depth of modulation (change in instantaneous firing frequency) was increased, as well as clear silences (lack of spontaneous activity) between sinusoids. Adapted from Ackerley et al (2017) [13]. (C) Sinusoidal movements of the foot were imposed when the participant either closed their eyes or watched their foot move. The depth of modulation (change in instantaneous firing frequency) was decreased when the participant had combined proprioceptive and visual input. Adapted from Ackerley et al (2019) [12]. All microneurography recordings were from Ia muscle spindle afferents originating in the extensor digitorum longus muscle of the leg. Horizontal scale bars = 1 s.

*

  *[13] R. Ackerley, J.M. Aimonetti, E. Ribot-Ciscar, Emotions alter muscle proprioceptive coding of movements in humans, Sci. Rep. 7 (2017) 8465. doi:10.1038/s41598-017-08721-4.

  Neurophysiol. 101 (2009) 633-40. doi:10.1152/jn.91041.2008. **[15] V. Hospod, J. Aimonetti, J.P. Roll, E. Ribot-Ciscar, Changes in human muscle spindle sensitivity during a proprioceptive attention task., J. Neurosci. 27 (2007) 5172-8. doi:10.1523/JNEUROSCI.0572-07.2007.

This work found that multisensory information can impact on the feedback from muscle afferents. Visual information was shown to interact with proprioceptive information, where muscle afferent activity increased when only proprioceptive information was available, thus providing increased feedback under unisensory conditions.

Funding: R. Ackerley is funded by a European Research Council (ERC) Consolidator grant under the European Union's Horizon 2020 research and innovation programme [grant agreement No. 772242].