Malika Ibrahim-Ouali

Frédéric Dumur

Steroidal chalcones and derivatives: A review

Keywords: chalcones, steroids, enones, pregnenolone

It was observed that the presence of heterocycles to steroids often leads to a change in their physiological activity and the appearance of new interesting biological precursors. Recent developments in the syntheses of steroidal chalcones and their derivatives are described herein. The biological activities of those steroidal derivatives for which data are available are given. The chalcone moiety shows great promise to allow us to develop novel molecules with characteristic functions.

Introduction

Steroids represent a large and important class of biologically active polycyclic molecules that are widely used in medicinal chemistry. [START_REF] Zeelen | Medicinal chemistry of steroids[END_REF][START_REF] Trager | [END_REF][START_REF] Biellmann | [END_REF] Even today, the total synthesis of steroids by new and more elaborate strategies continues to interest chemists. Thus, many total syntheses of biologically active steroids have been developed in recent decades. Interest in the total synthesis of such molecules continues to this day. 4,5 Chalcones (1,3-diphenyl-2-propen-1-one) are well known for their interesting biological properties. It is thus known that chalcones exhibit biological activities, such as anticancer, antioxidant, anti-inflammatory, 6,7 and antimicrobial activities. 8,9 Two phenyl rings and a , -unsaturated carbonyl system that connects them constitutes the structural base of chalcone. It has been shown that the nature of the substituents on the phenyl rings and their conformation in the central structure is at the origin of their biological activities. Some work has shown that reactions causing conformational changes, such as substitution on the double bond or epoxidation, can lead to a decrease in bioactivity. 10 The synthesis of hybrid compounds from the coupling of two or more natural products is at the origin of an inexhaustible new class of molecules with many structures. The objective of such syntheses is to combine the pharmacological properties of two or more biologically active molecules to increase or modulate these activities to obtain derivatives exhibiting new properties. 11,12 Previous works of literature have been reported that such structures as steroidal chalcones have been shown to bear a lot of different biological activities such as antimicrobial, anti-inflammatory, anticancer, antioxidant, and hypotensive activities. [13][14][15][16] This review presents the various syntheses of steroidal chalcones and their derivatives along with interesting biological activities, which have appeared in the literature in recent years. To the best of our knowledge and much to our surprise, there are no reports on this subject.

Synthesis of Steroidal Chalcones and Derivatives

In 2007, Negi et al. 17 reported the synthesis of steroidal chalcones from estradiol. Some of these compounds have significant anticancer activity against certain human cancer cell lines. Estrone 1 was chosen as the starting aldehyde substrate to carry out the synthesis (Scheme 1). Thus, to prepare the aldehyde derivative 5, a protection reaction by methylation of the phenolic hydroxyl of 1 was first carried out using dimethyl sulfate and anhydrous potassium carbonate in anhydrous acetone under reflux. Protected estrone 2 was isolated with a yield of 91%. After a reaction of reduction using sodium borohydride in a mixture of methanol-chloroform (2: 1), the ketone of derivative 2 led to the corresponding alcohol 3 in a yield of 94% and an acetylation reaction using acetic anhydride in dry pyridine of this latter, provided the steroidal derivative 17-acetate 4 in a very good yield. A Vilsmeier formylation reaction, 18 using dimethylformamide and phosphorus oxychloride at 80 ° C resulted in the aldehydic substrate 5. The desired steroidal chalcones deprotected at the C-17 position were obtained by reacting the substrate 5 with various acetophenone derivatives in a solution of hydroalcoholic potassium hydroxide at 7%. Chalcones 6 and 14 were then acetylated with acetic anhydride to give the corresponding acetylated derivatives 7 and 15. Scheme 1. Synthesis of steroidal chalcones 6-17.

One of the chalcone derivatives was further modified to the corresponding 1-indanone derivative 18 using the trifluoroacetic acid-catalyzed Nazarov cyclization reaction (Scheme 2). Indanone 18 was prepared in a yield of 23% by treatment in a sealed tube of chalcone 7 with trifluoroacetic acid for 4 h. 19 Some of these chalcones show significant anticancer activity against certain human cancer cell lines. This is the case with derivatives 9 and 18 which exhibit real activity against MCF-7, a hormone-dependent breast cancer cell line. Indanone 18 showed better activity than the parent chalcone against the MCF-7 breast cancer cell line. Using the erythrocyte as a model system, the most active chalcones were also tested for osmotic hemolysis. It has thus been shown that the fragility of erythrocytes is not modified by these biologically active chalcones. We can therefore consider these chalcones as non-toxic for normal cells. Scheme 2. Synthesis of the steroidal indanone 18 from chalcone 7.

In 2013, Sarma et al. 20 reported the synthesis of a new class of chalconoyl pregnenolones based on a Claisen-Schmidt reaction without solvent and under microwave activation conditions.

Cortisone, estrogen, testosterone, and progesterone among others are natural hormones biosynthesized from pregnenolone, a neurosteroid derived from cholesterol by biosynthesis in the adrenal gland and the central nervous system. 21,22 The condensation between the acetate derivative of pregnenolone 20 and the benzaldehyde 21 carried out without solvent and by microwave activation using 20 mol% of the catalyst, I2-Al2O3 led to the desired compound with a good yield of 86% after only 6 min of reaction at 250 W of microwave power. The authors were thus able to prepare sixteen new steroidal chalcones by condensation of compound 20 with different substituted benzaldehydes (Scheme 3). Furthermore, no by-product was observed, these clean reactions led to the desired chalcones with good yields of between 79 and 86%. Longer reaction times or an increase in the percentage of catalysts resulted in the decomposition of the desired product. In addition, the nature of the substituents on the aromatic ring of benzaldehyde does not affect the yield of the desired product.

Scheme 3. Synthesis of steroidal chalcones 22-37.

Two fungal strains Aspergillus niger and Candida albicans and two bacterial strains Bacillus subtilis and Escherichia coli were used to test the synthesized derivatives 22-37. Some have interesting inhibitory activity. Furthermore, it has been observed that this activity is considerably reduced if the double bond is epoxidized. The presence of the unsaturated chalcone moiety, therefore, plays a significant role in this inhibitory activity.

In 2015, El Sayed Aly et al. 23 described the synthesis of steroidal chalcones from cholesterol in good yield. The starting chalcones were synthesized as described in Scheme 4. Intermediates 40a-b and 41a-b were prepared in average yields at room temperature by propargylation of chalcones 39 using propargyl bromide and the potassium carbonate as the base. The chalcone 40c was prepared by the Claisen-Schmidt condensation reaction of intermediate 42, previously propargylated as described above, in the presence of pdimethylaminobenzaldehyde. In doing so, the extinction of propargyl bromide as a quaternary ammonium salt by the dimethylamino group is prevented which would not have been the case if the chalcone engaged had been propargylated as described previously for compounds 40a-b and 41a-b. Scheme 4. Synthesis of chalcones derivatives 40a-c and 41a-b.

Cholest-5-en-3β-ol 43 was activated as bromide in very good yield under Appel conditions, 25 which means treatment with CBr4/PPh3 to afford 3α-bromocholest-5-ene 44 due to inversion of the configuration at the C-3 carbon (Scheme 5). An SN2 substitution of the bromine atom of compound 44 with the N3 group was ensured by refluxing with NaN3 in dry DMF to afford 3β-azidocholest-5-ene 45 after inversion of the configuration again at the C-3 carbon. The product was isolated in good yield.

The target cholesterol-chalcone conjugates 46a-c and 47a-b were prepared by reacting 3β-azidocholest-5ene 45 with propargylated chalcones 40a-c and 41a-b under CuAAC conditions. 26 The reactions proceeded fairly in gently refluxing THF/H2O mixture containing L-ascorbic acid as a reducing agent and a catalytic amount of CuSO4•5H2O.

The chalcone modified cholesterol derivatives 46a, c, and 47b were active against E. coli. They were as active as ampicillin. Thus, the chalcones possessing unsubstituted phenyl, and p-dimethylaminophenyl, as well as 2thienyl alternatives were more active than other congeners against E. coli. The chalcone derivative 46c emerged as the most promising antimicrobial probe. It was as active as the controls against E. coli, S. aureus, and C. albicans.

In 2016, Liu et al. 27 described the synthesis of steroidal chalcones using the molecular hybridization approach.

The target molecules can be synthesized from the corresponding alkynes and steroidal azide 49 through click chemistry. The synthesis of the steroidal azide 49 is shown in Scheme 6. Bromoacetylation of DHEA under Schotten-Baumann conditions 28 afforded the corresponding bromide 1 in 87% yield, which then reacted with NaN3 in DMSO to give the steroidal azide 49 in 85% yield. Scheme 6. Synthesis of the steroidal azide 49.

The steroidal azide 49 was used directly for the next step after a simple aqueous work-up procedure. In the first step, a two-phase solvent system (DCM/H2O) was used; the base NaHCO3 in the aqueous phase neutralized the generated HBr, while the starting materials and the product were in the organic phase. Besides, this condition avoided unwanted side reactions of bromide 48 with H2O.

The synthesis of alkyne building blocks is shown in Scheme 7. Treatment of 4-hydroxy acetophenone and 4hydroxybenzaldehyde with propargyl bromide in the presence of K2CO3 gave compounds 50 and 51, respectively, which then underwent the KF/Al2O3-catalyzed Claisen-Schmidt condensations with aromatic aldehydes and acetophenones, affording substituted chalcones 52a-e and 53a-b. Scheme 7. Synthesis of alkyne building blocks 52 and 53.

With the azide and alkyne building blocks in hand, the steroidal chalcones 54 and 55 were efficiently synthesized through the standard CuAAC reactions in moderate to good yields (Scheme 8). These compounds showed moderate but remarkably decreased inhibitory activity against SH-SY5Y cells with IC50 values more than 36.64 M. They exerted weak or no activity toward other cancer cell lines regardless of their substituent patterns and the nature of substituents. Scheme 8. Synthesis of steroidal chalcones 54 and 55.

In 2020, Xiang et al. 29 reported the synthesis of a series of novel steroidal-chalcone derivates based on the molecular hybridization strategy.

The starting material was inexpensive pregnenolone acetate which was reduced by H2 to obtain compound 57. The intermediate 58 was acquired via a hydrolysis reaction. Subsequently, compound 58 was oxidized to yield the progesterone 59, whose acetyl group was then oxidized to generate the key intermediate 60. The compound 60 reacted with N-Boc-ethylenediamine to afford 61. The compound 61 was first deprotected and then reacted with various carboxylic acids to offer the final productions 62a-i (Scheme 9). The synthesis of target compounds 64 began from the intermediate 61 that was dehydrogenized by DDQ to form the intermediate 63.

Then, the final compounds 64a-i were obtained through deprotection and condensation reaction. Scheme 9. Synthesis of steroidal chalcones 62 and 64.

A preliminary biological activity evaluation was carried out. The antiproliferative results showed that most compounds exerted weak activity against human colon cancer cells HT-29 and lung cancer cells A549. In contrast, most compounds showed excellent growth inhibitory activity against human TNBC cells MDA-MB-231. Among these compounds, compounds 62c (with R1 = OMe and R2 = R3 = R4 = H) demonstrated the most potent activities with the IC50 values of 0.42 μM. Moreover, compound 62c could accumulate the ROS levels in MDA-MB-231 cells and induced cell apoptosis. Western Blot analysis documented that compound 62c could downregulate anti-apoptotic Bcl-2 but up-regulate pro-apoptotic Bax expression. Additionally, compound 62c could also activate the caspase-3 signaling pathway, which accelerated the apoptosis of MDA-MB-231 cells.

In 2021, Rashid et al. 30 described the synthesis of chalcone derivatives of pregnenolone via base-catalyzed Claisen-Schmidt condensation reaction.

The synthesis of compounds 66a-c was carried out in ethanolic potassium hydroxide by treating Pregnenolone 19 with a series of both the substituted and non-substituted aromatic aldehydes 65a-c. The synthetic process is outlined in Scheme 10.

Scheme 10. Synthesis of chalcone derivatives of pregnenolone.

Subsequently, the syntheses of 3,4-dihydropyrimidin-2(1H)-one 70a-c and 3,4-dihydropyrimidin-2(1H)thione 71a-c and 2-amino-3,4-dihydropyrimidine 72a-c derivatives of pregnenolone-chalcones were carried out in ethanolic potassium hydroxide by treating chalcone derivatives of pregnenolone 66a-c with urea 67/thiourea 68/guanidine HCl 69. The process of synthesis is outlined in Scheme 11.

Scheme 11. syntheses of 3,4-dihydropyrimidin-2(1H)-one 70a-c and 3,4-dihydropyrimidin-2(1H) -thione 71a-c and 2-amino-3,4-dihydropyrimidine 72a-c derivatives of pregnenolone-chalcones.

Cell viability test of synthesized steroidal chalcones and their pyrimidine and thiopyrimidine derivatives against human breast (MCF-7), human lung (A549), and human prostate (PC-3) cancer cell lines was done using MTT ((4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), assay. Compounds were further evaluated for their inhibition potential against recombinant human DHFR (rhDHFR). All compounds showed activity from the low micromolar to the submicromolar range. Compound 72b with an IC50 value of 180 nM emerged as the most potent compound against rhDHFR. Interaction of the newly synthesized pregnenolone derivatives with hDHFR and estrogen receptor alpha (ERα) was also explored via docking simulations. The overall results of hDHFR inhibition have shown that these analogs can be further optimized and developed as potent anticancer agents.

Conclusions

The present review offers up-to-date literature on the syntheses of steroidal chalcones and derivatives reported during the last years. An almost infinite quantity of new molecules with various structures can be obtained by coupling two or more natural compounds. The objective of such syntheses is to combine the pharmacological properties of two or more biologically active molecules to increase or modulate these activities to obtain derivatives exhibiting new properties. In general, it has been observed that the unsaturated chalcone moiety in the synthesized compounds is at the origin of the activity of steroidal chalcones. Generally speaking, it is observed that the steroids and the resulting derivatives continue to be of interest to researchers, given the biological potential inherent in this family of molecules.

© ARKAT USA, Inc

© ARKAT USA, Inc Scheme 5. Synthesis of derivatives 46a-c and 47a-b.

Acknowledgements

The CNRS and the Ministère de l'Enseignement Supérieur et de la Recherche have financially supported this work.

Authors' Biographies


Jean Michel Guillot
email: jean-michel.guillot@mines-ales.fr

Adaptations of Olfactometry and Odour Measurement with COVID-19 Crisis

The year 2020 was characterised by a worldwide pandemic crisis due to dissemination of a Coronavirus, the SARS-CoV-2 also named COVID-19. Many economic and social domains were impacted by adaptions or restrictions by the objective to limit local dispersion and more global flow of the virus. Without describing all human health effects of this virus, one characteristic is temporary anosmia. In this paper, firstly the physiological impact is synthetized to explain the anosmia. Secondly, because olfactometry need human smell, adaptations of sensorial methods are described. These adapted methods were proposed by laboratories/companies in charge of olfactometry or odour measurement and mainly concern how the distance guarantees between panellists were proposed.

Introduction

The aim of this paper is to summarize two aspects of olfactory impact of COVID-19 crisis. The first part concerns the short description of the physiological impact taking into account that many studies are in progress to study mechanisms of action of this virus on sensorial receptors. Then it is just a synthesis of scientific results or hypothesis known at the beginning of year 2021. The second part, more developed, is dedicated to technical measurement of odours. Because olfactometry and sensorial odour measurement are based on direct sniffing with human nose, panellists cannot carry out tests with a mask. It means, by removing the mask, they are not protected during experiments if the area or people around could be contaminated. In order to limit risks, laboratories proposed adaptions of their protocols. By considering health of panellists as a priority, laboratories were able to adapt their equipment and to propose measurements in accordance with standards or reference methods.

Physiology and anosmia caused by COVID-19

The global mechanism of olfaction is a result of chemosensation. Even if humans, because of their evolution, are less sensitive and less dependent on chemical signals than many mammals, olfaction is still an important sense for both environmental assessment (potential hazard detection with unknown odour) and wellness or pleasure (perfumes, food…). So, losses of olfaction capacities are impacting strongly quality of life. Such an impact could be limited on time in case of temporary olfactory dysfunction. Anosmia is one of existing trouble that change day life because it creates a lack of perception of the environment. This anosmia is generally due to infection/irritation of nasal cavity. Many infections can explain such a disorder like cold, flu but also sinus infection, allergic or no-allergic rhinitis, smoking impact… Of course, the stuffy nose can be a consequence of a mechanical trouble for nasal flow that could be limited by polyps or tumors and then decrease the capability to smell. But, the loss of smell with COVID-19 infection is more complex and more in relation with physiological impacts. The Figure 1 tries to represent both aspects of olfaction structure: the nasal cavity where airflow is distributed from external atmosphere to olfactory neuroepithelium and neuroanatomic connections of the olfactory nerve that illustrates the complexity of signal treatment. With such a figure, it's easy to understand than a small infection affects normal airflow distribution and the access to epithelium and, that if the epithelium is a gate for a stronger infection, consequences are wider.

Olfactory perception can be affected by SARS-CoV-2 (COVID-19) infection. If seasonal infections like flu are known to cause temporary anosmia, it seems that COVID-19 has a more complex action. Many studies showed that the virus can affect the olfactory receptor area without major other impact, or that this area could be a gate for infection of other human parts like brain and then creating headaches, memory and concentration problems… The following paragraphs 2.1 and 2.2 describe respectively the classical anosmia with a virus like flu and the complexity of smell alteration with COVID-19. It is important to note that COVID-19 actions are under study and that some results must be considered as hypothesis. Anosmia is not the only dysfunction of olfactory perception and other troubles are also defined [START_REF] Patel | [END_REF] and listed in Table 1. If anosmia is a characteristic of some infections, it exists three etiologic categories [START_REF] Patel | [END_REF]. These categories are synthetized in Table 2.

Classical anosmia with flu

With a cold or a flue, affected people are often concerned by a temporary anosmia. Such a condition is limited on time, generally few days and not few weeks. The global mechanism is congestion of the nose (stuffy nose and congestion around olfactory epithelium, the location of first smelling action). The presence of viruses in the nose is very common and partially linked to seasonal diseases like flu or cold.

Anosmia with COVID-19

The mechanism is more complex with COVID-19 and the impact on smelling is clearly different. Firstly, anosmia is one of the main symptom of covid-19 infection. Secondly, anosmia duration is really increased comparatively to a cold. This trouble is observed for few weeks and sometimes up to 2 months or even more.

Globally, the come back to normal situation could take long time both for the sensitivity and for quality. With strong olfactory affection, some people must restart learning to be able to characterise and to distinguish odours.

In fact, with COVID-19, it's not just a stuffy nose trouble but a more global impact. The virus attacks the olfactory epithelium in the nasal cavity and the olfactory bulb, but some studies are also focused to understand how the nervous system could also be affected [START_REF] Ikonomidid | Anosmie et covid-19[END_REF]. With this crisis, the COVID-19 olfactory dysfunction is then an active research domain in terms of physiology and physiopathology. With SARS-CoV-2 infection, potential mechanisms of olfactory disorders are studied and among all studies, potential receptor of the virus are particularly studied like ACE-2 receptors.

Technical adaptations for sensorial odour measurement with COVID-19

Three cases of adaptation are described depending on how sensorial odour measurement is carry out. These three sections concern dynamic olfactometry, measurement of intensity and/or quality in laboratories and intensity or sniffing outside.

All the descriptions in paragraphs 3.1 to 3.3 depend on two main parameters:

• Panellists cannot wear a mask for sensorial odour measurement to avoid filtration effect of the mask. Even if masks used for virus trapping do not change the chemical composition of the atmosphere. The mask itself can give a weak odour background incompatible with direct measurement with human nose. • Without mask, a distance must be kept between panellists or a physical protection must limit air exchange between close people.

Adaptations for dynamic olfactometry

For measurements by dynamic olfactometry according to EN standard (CEN, EN 13725:2003,), protocol is described about air quality of the room where tests are carried out. This protocol concerns room temperature, humidity level and odourless atmosphere. This last point could be guaranty by filtration of ventilated air on active charcoal, interdiction to enter with odorous materials, perfumes… At all these requirements, COVID crisis implies to be sure that ventilation or air conditioning cannot introduce the virus in the air or typically avoid the diffusion due to air recycling. So at the odourless requirement, the virusless atmosphere is added as objective.

The use of hydro-alcoholic solutions for disinfection must be well defined. Firstly, the solution must be as odourless as possible (some commercial products are odorous), the use on hands of panellists is practiced outside the room for olfactometry and few minutes could be necessary to verify that no residual odour is present.Because panellist could be asymptomatic in terms of olfaction loss, other parameters like fever can be controlled easily before a measurement session in order to eventually exclude panellist with fever.

After considering the environment (room), panellists, a specific attention about the equipment (olfactometer) is necessary. It's obvious to clean parts that be touched by panellists at each session such as buttom to give an answer both for forced choice or Yes/No methods. Independently to cleaning and depending on the structure of the olfactometer and the place, two approaches were developed:

• A panellist has generally a seat quite close to his/her neighbour panellist. The separation plates that isolate panel members around each individual places, are commonly not very large. So the first option was to place larger separation plates (for examples made with polymers such as Plexiglas, polycarbonate…). The objective is simple and based on the principle of isolation of members in a limited area. On the same waaay to increase to distance, an olfactometer with 6 places can be uses with only 4 people and free places in the middle. If this technique must applied for olfactometers with 4 places, it implies to propose 2 identical rounds with each time only 2 panel members. This last description is sample and time consuming • The second approach was to increase distance between individual sniffing ports of the olfactometer. In that case, it implies to have longer tubing between dilution into the olfactometer and the sniffing port. With this approach, the volume of this new tubing and flow time must be calculated in order to adapt the duration for exposure (stronger volume to flush) and for voting (stronger delay to smell the proposed dilution). Of course, combinations of these two approaches can be developed but a choice for one option is enough if all rigour around is applied.

Adaptations for measurement of intensity and/or quality in laboratories

For intensity and/or quality measurements of emitted odour (from food, materials…), it exists a lot of protocols where panel members have to describe the measured parameter. For such a situation, a specific room is dedicated to place panellists in clean and quiet conditions. All aspects about the room that were developed at the beginning of paragraph 3.1 are also available for this kind of measurements. All remarks about panellists are also the same for the period of time before the test like disinfection with hydro-alcoholic solution and potential impact on smelling. In that case, two aspects must be considered:

• The members cannot smell the same sample placed in the same flask, members after members. It implies to prepare a least as many samples than panellist number. The difficulty is to propose strictly similar samples (weight, size, composition…) in a similar container (flask…). It's easy for homogenous material to be tested but more difficult if not.

•

Depending on surface/place into the room, it can be recommended to let just one panel member in the room, then another one… or two members placed at opposite sites into the rooms, then, new members… The method is then adapted depending of the number of members involved in the sniffing test, the initial capacity of the room and the choice of limited capacity (divided by 2 or 3). The examples of capacity evolution into a room is shown in Figure 2. These examples are for 6 panellists that must smell identical samples in a room designed for the odour test. Of course, such an example must be adapted with the room configuration and the total number of panellists for the selected test. The cleaning of the room is carried out after the passage of the 6 panellists. Two main limitations of adaptation must be considered. Firstly, the increase of experimental time because panellists cannot smell strictly at the same time and by limiting the occupation and avoiding meeting of members at the door of the room, the experiment duration is quickly multiplied. Secondly, because it exists a delay between the first member and the last one, the tested product must be stable for such a period. If not, the preparation must be scheduled as a function of sniffing period to let strictly the same delay from conditioning ant sniffing test. For quality measurements involving a very regular training, the lockdown in many countries was sometimes incompatible with the maintain of such ability as defined by protocols and frequencies or learning/verifications. Then, in some case, it was necessary to verify if trained human noses were efficient enough to distinguish and characterise odours after few weeks without tests. In case of low performance, it was necessary to restart intensive training. Logically, adaptations for outside are very low comparatively to measurement in a laboratory. So, this paragraph must be considered as more informative than strong adaptations. That is the reason why it concerns mainly recommendations on how to work outside during the pandemic period. For the determination of odour in ambient air by using field inspection, the standards (CEN, EN 16841:2016, part1 and part 2) describe methods to smell alone in the environment.

For the grid method (part1), the panel member is alone on one point. Because he/she is alone, it's possible to smell without mask at the defined point for a 10 minutes duration. Of course, it could be decided by temporal regulation that people must wear a mask and that is forbidden to stay outside due to lockdown or allowed only some parts of the day out of curfew. Depending on the study, it seems possible to be allowed to carry out such an experiment. A limitation could be if the smelling point is placed in a zone where a lot of people are moving like the sidewalk at the crossroads of two streets and then the safety of the panel member could be affected by smelling without mask protection.

For the plume method, if the member is also alone or enough separated on time with others, the experiment can be easily carry out in the countryside. The same restriction than for grid method can be reported: conformity with temporal and exceptional regulation, no possibility to remove the mask in areas with many people around. After this practical aspect about authorization to remove a mask or not depending on the location, the question of representative activity must be considered because of abnormal situations in some economic sectors. On one hand, it can be considered that such experiments are not representative of real activities when economic sectors are stopped (example: a factory closed) or at an extremely low level of activity (low production and then probably low emissions and low exchanges with transport decrease). But, on the other hand, it's also an opportunity to characterise an area and suspected source impact with a lower air pollution background. In all cases, experiments can be carried out taking into account the representativeness of the period.

The same remarks are available with the use in the environment of direct dilution devices such as "field olfactometers". It is important to note that if such a field device is used in this pandemic period, it's a way to afraid people looking at the panel member. The dilution device could be assimilated, as a filtration system because atmosphere could be potentially hazardous. Unfortunately, this kind of reaction exists even in normal situation when a panellist is observed with a device on her/his face because it's questioning for population. So, in a crisis period with atmospheric contaminants, with unpredictable reactions of people around, the device cannot be used as usual without thinking of potential reactions. In order to avoid panic, the measurement with a "field olfactometer" must be thought depending on the situation and of course with a communication and explanation process to avoid misunderstanding.

Conclusions

Since the end of year 2019 and the beginning of year 2020, the COVID-19 caused a worldwide crisis. One human characteristic of this virus is the high capacity to lead to anosmia for few weeks. Unfortunately, stronger impacts on human health with severe respiratory conditions are the worst aspect of this pandemic. With this infection, even if the frequency of anosmia is high, it's not important comparatively to severe case and death rates and long term affections. Many economic sectors were highly impacted or sometimes totally stopped. The olfactometry and odour measurements were indirectly affected. Of course, variations in the economic sectors influence the activity of laboratories on odours. This variation for the number of odour measurements can change on both directions because less activities leads to a decrease of checks (when its directly linked to the activity) or increase the opportunity to characterize some aspects that are not studied during a full charge of activity and busy periods.

It is a key point to mention that laboratories and companies, where some tests are based on human smelling, demonstrated abilities for adaptations combining quickness, safety and global efficiency. So, of course, COVID-19 crisis had an impact on sensorial odour measurements but the proposed adaptions were efficient to continue experiments in major cases.

Figure 1 :

 1 Figure 1: Complexity of olfaction with neuroanatomic connections of the olfactory nerve by Pinto (2011).

Figure 2 :

 2 Figure 2: Examples on how to place six panellists as a function of the decided room capacity for measurement of odour quality (Each place is used once by only one panellist and the capacity dividing factor defines the number of consecutive partial tests to reach the global result).

Table 1 : Other olfactory problems than anosmia

 1

	Desease	Dysosmia	Parosmia	Phantosmia
	Description	Difficulty	Sensation	Sensation of an odour
		to identify an odour	of a different odour	without real presence

Table 2 :

 2 Three categories of etiology for anosmia

	Problem	Conductive losses	Sensorial (local)	Sensorial (central)
	Cause	Obstruction	Olfactory	Central nervous system
		of the nasal way	neuroepithelium damage	disease

Acknowledgments

Laboratories and companies in charge of dynamic olfactometry and/or sensorial measurement based on smelling were reactive to imagine and carry out adaptions compatible with their equipment.


Laetitia Majoli

Alexis Evstratov
email: alexis.evstratov@ema.fr

Jean-Michel Guillot

Jean-Franc ¸ois Chapat

Jean-Luc Le Loarer

Intensive pyrolysis of unsaturated organic precursors over acidified oxide surfaces

Keywords: Alumina, Non-catalytic heterogeneous pyrolysis, Acid-base surface properties, Adsorption, Desorption, Thermal stability

Carbon-like composite materials fabricated by non-catalytic heterogeneous pyrolysis (NCHP) of organic precursors over porous oxide supports are used in a variety of fields. The intensification of NCHP procedures for fabrication on a commercial scale is technically problematic. Direct intensification of operation parameters cannot be considered as the most effective method. For instance, an increase in support bed temperature often takes the pyrolysis process into the homogeneous reaction field, and greater gas space velocities and initial organic precursor concentrations lead to a rise in operating costs because of considerable losses of raw materials and increased power requirements.

This paper describes an attempt to intensify non-catalytic heterogeneous pyrolysis procedures by optimizing the acid-base properties of the reaction agents (supports, precursors).

A procedure of controlled acidification of the alumina support followed by thermal degradation of surface base salt was applied in order to cover the support surfaces with additional Lewis acid sites functioning as efficient host centres for precursor molecules. Unsaturated organic compounds, powerful Lewis bases, were used as the precursor agents.

A probable elementary mechanism for non-catalytic heterogeneous pyrolysis of unsaturated organic precursors over an oxide surface reinforced by Lewis acids is proposed. The main characteristics of the composite hydrophobic materials produced by the NCHP technique using pre-acidified alumina samples-their chemical compositions, surface affinities to water, thermal stabilities in air, dynamic VOC adsorption and desorption capacities are presented and discussed. The elaborated composites are non-flammable in air until 600 8C, whereas the thermal stability limit of activated carbons is situated in the 250-300 8C range. As compared to activated carbons, these materials manifest really stable surface activities in VOC adsorption-desorption cycles.

Introduction

Products of the non-catalytic heterogeneous pyrolysis (NCHP) of organic precursors over the surfaces of porous oxide supports are used as composite adsorbents [1], tyre fillers [2], catalyst carriers [3] and in some another technical fields. The particular features of the application of this technique to the modification of hydrophobic mineral supports are widely discussed in the literature [4][5][6][7][8][9][10][START_REF] Majoli | Adsorbants Alternatifs de Compose ´s Volatils, 20e `me Re[END_REF].

Intensifying the NCHP procedures is a major technical problem. In order to increase the efficiency of the process, it seems logical to explore, first of all, the avenue of intensifying the operation parameters. Among the main operation parameters of NCHP, the following can be identified as important: the temperature of the mineral support bed, the gas flow rate and the initial concentration of the organic precursor.

However, this type of modification is rather limited in its potential efficiency. Concerning the thermal factor, increasing the bed temperature often takes the pyrolysis process into the homogeneous reaction field [START_REF] Bogomolov | [END_REF]. Due to the kinetics of the procedure, exceeding certain limits with regard to the gas flow rate and initial concentration of organic precursor leads to a less efficient precursor utilization and losses of inert gas carrier, thus causing a general increase in operating costs.

An alternative technical solution for intensifying the NCHP procedure probably consists in optimizing the chemical properties of the reaction medium. The evolution of an oxide matrix during any common NCHP procedure can be presented as three elementary steps, shown in Fig. 1. After the restoration of its initial state by means of dehydration reactions, the oxide surface acquires electrically charged sites named active Lewis centres. Positively charged sites, Lewis acid (LA) sites, are able to act as reception centres for organic molecules, which generally manifest Fig. 1. Probable elementary mechanism of NCHP procedure.

Lewis base (LB) properties because of their well-known reduction capacities. Lewis-type acid-base interactions within a dynamic ''organic precursor oxide matrix'' system lead to the formation of transition complexes with weakened carbon-hydrogen bonds. This bond weakening is caused by the redistribution of electron densities between the precursor organic molecules and the Lewis acid sites involved. Further, thermal destruction and/or partial oxidation of the transition complexes transforms them into carbonized structures chemically bonded to the support surface. As a rule, because of incomplete dehydrogenation of transition complexes, the chemical compositions of the carbonized surface structures correspond to the general formula C n H m , where n ) m. NCHP products with maximal n/m ratios for their carbonized surface layers are considered to be composite materials of the best quality (high thermal stability, considerable hydrophobic surface capacities, etc.).

So, reinforcement of the Lewis-type acid-base interactions within the reaction medium seems to be a promising alternative solution for intensifying the NCHP procedure. This measure is expected to be efficient both for promoting transition complex formation and for further degrading these intermediate structures to carbonized surface products owing to stronger interactions between organic precursor molecules and LA surface centres followed by complementary weakening of carbon-hydrogen bonds.

Unsaturated organic precursors (alkenes, alkynes) can be successfully used as extremely powerful Lewis bases. With regard to oxide matrices, in particular activated alumina supports, their Lewis acidity may be considerably reinforced by means of controlled treatment in inorganic acid solutions followed by thermolysis of the salt layers formed on the surface. This procedure apparently results in a number of new Lewis acid sites over the acidified oxide surface (AlAn x , Al(OH) y An z ! Al n+ ).

Experimental

Modification of the surface properties of the activated alumina

The Lewis acidity of activated aluminas was increased by impregnation in a hydrochloric acid solution followed by thermolysis of the salt layers formed on the surface [START_REF] Majoli | Adsorbants Alternatifs de Compose ´s Volatils, 20e `me Re[END_REF]. Activated alumina (50 g) was placed in HCl solution (0.1 mol L À1 ) for 1.5 h. After this treatment, the new support was washed four times and then dried in ambient atmosphere for 24 h.

Conditioning of original oxide supports

The mineral support (15 g) was placed in a 25-mm diameter tubular stainless steel reactor (experimental device shown in Fig. 2). It was heated in a furnace over a temperature range between 500 and 900 8C, with an air flow (1.2 L min À1 ). The gas space velocity was equal to 9300 h À1 [START_REF] Majoli | Adsorbants Alternatifs de Compose ´s Volatils, 20e `me Re[END_REF]. The evolution of the humidity content was monitored using a Dostmann P600 electronic probe.

The original adsorbents were activated aluminas. Their pore structure parameters, as well as the surface affinities to water vapour are given in Table 1. The pore size distributions for initial supports are shown in Fig. 2.

Preparation of hydrophobic adsorbents

Synthesis of carbon-mineral adsorbents was carried out using the chemical vapour deposition (CVD) method according to the NCHP procedure. Firstly, mineral supports (10 g) were heated over the temperature range 500-900 8C, in air flow (1.2 L min À1 ) for 3 h (internal diameter of reactor 25 mm). Secondly, the support was exposed at the same temperature to a 70 mL min À1 flow of inert gas charged with an olefin-type organic precursor. The precursor concentration was 190 g m À3 . The device shown in Fig. 3 was used for this operation.

Material characterization 2.4.1. Acid-base properties

The acid-base properties of the alumina supports, both initial and after treatment with acid solutions, were characterized according to the analytical method proposed by Tanabe [START_REF] Tanabe | Solid Acids and Bases[END_REF] and illustrated in Fig. 4. This method enables the transformation of Lewis active sites to Broensted active sites over different mineral surfaces to be checked by means of the heterolytic water dissociation reactions:

(I) Lewis base to Broensted acid transformation: M- O À + H 2 O @ M-OH + OH À ; (II) Lewis acid to Broensted base transformation: M + + 2H 2 O @ M-OH + H 3 O + .
The method consists in putting a powdered oxide sample in contact with distilled water. The heterolytic dissociation of water molecules caused by their interaction with surface Lewis active sites, according to the reactions mentioned above, results in the appearance of additional hydroxyl and hydroxonium ions in the reaction medium. Basification of the medium thus corresponds to a predominance of Lewis base sites over the solid surface: the hydroxyl ion concentration is greater than that of hydroxonium ions. Conversely, acidification of the medium indicates a predominance of Lewis acid sites.

Curves I and II in Fig. 4 correspond to the reactions mentioned above.

Pore properties

Pore volume measurements were based on mercury pore distribution and carried out using a Micromeretics Autop-ores III. The specific surface was determined using a Micromeretics ASAP 2400.

Hydrophobic properties

The static water adsorption capacities (E 0,6 20 8C ) over the surfaces of the initial supports and hydrophobic composite adsorbents were determined. The support was placed in a 20 8C atmosphere, at 60% of relative humidity, and the mass increase determined until a stable value was reached.

Amount of carbon-like product quantities in hydrophobic composite materials

The quantities of carbon-like products deposited over the surfaces of the initial support were determined using a Horiba Emia 820 instrument.

Composite material behaviour under thermal treatment

TG/DTA analysis were carried out using a SETARAM 92 ATD model over a temperature range of 25-800 8C in air atmosphere. The heating rate was fixed at 5 8C min À1 and the sample mass was 35 AE 1 mg. The content of carbon for each composite sample was e 9 AE 1%, in order to avoid any significant influence of the quantitative composition of the material on the measured values. For the activated carbon sample, the carbon matter content was approximately 98%.

VOC adsorption capacity and material regenerability.

The adsorption capacities over the surfaces of an activated carbon (reference product) and hydrophobic composite materials were evaluated by means of dynamic adsorption tests with acetone as a test compound in dry air. The space velocity was 2000 h À1 (measured under standard conditions) and the sample mass was fixed at 10.0 g. The initial acetone vapour concentration was 650 AE 50 mg m À3 . Analyses of the inlet and outlet gas flows were carried out using a COSMA Graphic 730 flame ionisation detector (FID). Desorption tests were carried out with an air flow of 500 mL min À1 , while heating the adsorbent layer from ambient temperature to 200 8C. Fig. 3. Experimental device for support conditioning and composite material synthesis.

Results and discussion

Modification of the acid-base properties of the initial alumina supports

According to the reaction scheme for the elementary mechanism of the NHCP procedure (Fig. 1), acid-base interactions on an initial carrier surface can be intensified in the presence of powerful cationic acceptor centres (LAcentres). An original method was developed to increase the number of such active sites on the surfaces of the alumina samples. The alumina samples were processed in mineral acid solutions to cover their surfaces in a thermally unstable salt layer, which could be easily destroyed by heating, without any significant changes to the initial macroscopic structure of the bulk material. A generalized scheme of this procedure is shown in Table 2.

Among the aluminas tested as potential mineral matrices for hydrophobic composite materials (Table 1), carrier SPH 538E has the lowest Lewis basicity (Fig. 5), so can be considered a priori as the most promising material for altering its surface acid-base properties. This property, together with the well-developed porous structure of this activated alumina (formed especially of meso-pores, which are known to be more suitable than micro-pores for pyrolysis procedures), enabled SPH 538E to be chosen as a model substrate for producing a hydrophobic composite material using pre-acidified oxide supports.

The results, presented in Fig. 6, show that the acidthermal processing of an activated alumina leads to a complete alteration of its initial acid-base properties by supplying the surface with additional powerful active acceptor centres. Varying the technological parameters of the acid processing (treatment time, mineral acid concentration in solution), it is possible to manage the acid-base surface properties over a wide range of their eventual evolution (Fig. 7).

The acid-thermal processing results, at the same time, in profound changes to the porous structure of the alumina samples, producing a considerable development of their specific surface and increasing their total pore volume. New LA-centres placed over an acidified surface also increase its hydrophilic capacities by means of a strong donor-acceptor linkage of water molecules: Al + + H-OH ? Al-OH + H + . Data concerning the modification of the structural characteristics and water adsorption capacities of the SPH 538E alumina after acid-thermal processing of the sample for Fig. 4. Characterization principle of sample acid-base properties by pHmetric measurements (according to method described by Tanabe [START_REF] Cloirec | Les Composes Organiques Volatils Dans L 0 Environnement[END_REF]). 3 (results before pyrolysis process).

Hydrophobic behaviour of composite material under thermal treatment

The aim of the preliminary acid-thermal support treatment is to intensify the deposition of a carbon-like products over the carrier surface. The data presented in Table 3 confirm that acidified matrices are more active than unconditioned ones (sample 1-initial activated alumina after pyrolysis with an olefin-type precursor, sample 2activated alumina after acid treatment followed by thermolysis of the surface salt products and pyrolysis with the olefin-type precursor).

Assuming that all the new LA-centres have the same electronic structure (Al n+ ) and therefore manifest similar acceptor abilities, it is possible to estimate their relative concentration increase for the treated surfaces. For instance, for the SPH 538E alumina sample processed for 1.5 h in 0.1N HCl solution, this increase is around 3.7 times as compared with the initial alumina sample (Table 3, sample 1). Globally, the activity of acidified alumina samples in their interactions with unsaturated hydrocarbons is about 30-40% greater than for the non-treated templates (Fig. 8). The hydrophilic capacities of the composite materials created over acidified supports are also lower (Fig. 9).

The exceptional thermal stability of the carbon-like products formed over the acidified alumina surface after acid-thermal conditioning should be particularly mentioned. Differential thermo-gravimetric analysis (DTA) data (Fig. 10) show the great difference in behaviour between high-quality activated carbon (trade mark G90, French fabrication, curve 1) and hydrophobic composite materials (created by the NCHP procedure using different oxide supports, curves 2-4).

For the composite species tested during the DTA investigation their thermal oxidation resistances are in the following order: Activated carbon < 13X composite < initial SPH 538E composite << acidified SPH 538E composite.

The thermal oxidation resistance values are also in good qualitative correlation with the Lewis acidity of the mineral support surfaces.

Indeed, the surface of the well crystallized synthetic molecular sieve 13X is practically free from powerful Lewis active centres, both acid and basic type. An evolution of the pH level in a dynamic ''synthetic molecular sieve 13X/ water'' system appears to be extremely slow. This fact confirms the incapacity of such a material to favour heterolytic water dissociation reactions, due to a low number of aprotonic active sites over its surface. In this case, the carbonized surface structures produced by non-catalytic heterogeneous pyrolysis of hydrocarbon molecules must be relatively rich in hydrogen (see Section 1). Their reduction capacities expressed in terms of resistance to oxidation must therefore be rather high (Fig. 10, curve 2). Conversely, the structural disorder of amorphous surfaces leads to strong aprotonic site formation. Although the acidbase properties of the initial SHP 538E sample show weak Lewis basicity, it would be reasonable to interpret this fact in terms of the relative dominance of Lewis base sites over acid ones. The hydrophobic composite produced over the surface of the initial SHP 538E is more difficult to oxidize in air (Fig. 10, curve 3). The chemical composition of its surface layer is probably richer in carbon that in the case of hydrophobic composite materials produced over crystalline surfaces.

The better oxidation resistance of the composite created over the acidified alumina surface (Fig. 10, curve 4: no inflammation was observed until 600 8C) apparently corresponds to the chemical composition of its surface layer, which is highly carbon-enriched. The strong acceptor capacities of an acidified alumina surface lead to efficient degradation of transition complexes into carbon-like products (see Section 1).

The extremely high oxidation resistance of this type of hydrophobic composite is a very important element in any successful application of these materials to cyclic adsorption processes carried out according to the technological scheme of in situ ''adsorption-thermal regeneration''.

Hydrophobic composite material activities in adsorption-desorption cycles

Figs. 11 and12, together with Table 4, give comparative adsorption-desorption characteristics for a high-quality activated carbon G-90 (from Carbio-12 Ltd., France) and for hydrophobic composite materials over acidified SHP-538E alumina.

Concerning VOC adsorption, the initial capacity of the activated carbon exceeds that of the hydrophobic composite materials (test compound -acetone). At the same time, these materials demonstrate an ability to preserve their adsorption capacities in cyclic use when the adsorption step is followed by ''soft'' thermal regeneration in situ (Fig. 12), whereas the charcoal is progressively deactivated under the same operational conditions (Fig. 11).

In spite of considerable differences in their specific surface values and porous structures, the adsorption capacities of the two types of adsorbent defined under optimal aerodynamic conditions are rather similar (Table 4). Aerodynamic adsorption conditions for adsorption procedures are considered to be optimal, when the adsorption bed length h is greater than its minimal value h 0, allowing the adsorption front to be formed inside the bed, without any breakthrough of gas species to be adsorbed (h > h 0 ).

The ratio between the specific surfaces of the activated carbon and hydrophobic composite sample (Table 4) is 5.8 in favour of the first. Moreover, microporous materials, activated carbon included, are considered to be the most suitable adsorbents for VOC treatment procedures [START_REF] Cloirec | Les Composes Organiques Volatils Dans L 0 Environnement[END_REF][START_REF] Blocki | [END_REF]. However, the adsorption capacity of the mesoporous hydrophobic composite materials supported on acidified aluminas is not less than 25% that of activated carbon. This last fact shows that the adsorption ability of an elementary surface unit of the hydrophobic composites is greater than that of activated carbon.

Conclusion

Non-catalytic heterogeneous pyrolysis of unsaturated organic precursors over oxide supports can be considerably intensified by Lewis acidification of the support surfaces.

The Lewis acidification procedure for an activated alumina used as a model oxide support is simple, reliable and well controlled. A mineral support, with a mesoporous structure was chosen after acid-base characterization. After the acidification procedure, a sample of the hydrophobic composite, which contains up to 27% carbon as deposited mass, was produced.

Preliminary Lewis acidification of mineral supports enables the production of hydrophobic composite materials with high adsorption and thermal regeneration capacities and outstanding thermal resistance (no inflammation in air until at least 600 8C).

Fig. 2 .

 2 Fig. 2. Pore size distribution in initial activated aluminas.

Fig. 5 .

 5 Fig. 5. Acid-base properties of initial support.

Fig. 6 .

 6 Fig. 6. Comparison of acid-base properties of initial and acidified alumina SPH 538E.

1. 5 h

 5 in 0.1N hydrochloric acid solution are shown in Table

Fig. 7 . 5 a

 75 Fig. 7. Influence of impregnation time and acid concentration upon acidbase properties of activated alumina.

Fig. 8 .Fig. 9 .

 89 Fig. 8. Evolution of carbon mass concentration over time during NCHP procedure.

Fig. 10 .

 10 Fig. 10. Comparison of thermal behaviour for different adsorbents.

Fig. 11 .

 11 Fig. 11. Evolution of adsorption capacity of activated carbon over three adsorption-regeneration cycles.

Fig. 12 .

 12 Fig. 12. Adsorption efficiency of hydrophobic composite adsorbent supported on acidified alumina over four adsorption-regeneration cycles.

Table 1

 1 Characteristics of initial activated aluminas as potential supports for carbon deposition

	Aluminas	Specific	Total pore	Static water adsorption
		surface,	volume,	capacity, E 0.6 20 8C
		S BET (m 2 g À1 )	V P (mL g À1 )	(mass%)
	AA 1.5-3	303	0.393	19.4
	SPH 1515	169	0.718	8.4
	SPH 538 E	250	0.684	13.3

Table 2

 2 AlAn 3 + H 2 O ? Al(OH)An 2 + HAn Al(OH)An 2 + H 2 O ? Al(OH) 2 An + HAn

	Reactions on activated aluminas during their acidification process
	Step	Operation	Reactions	Product
	1	-	-	Activated alumina
	2 Al[(OH) 3 Surface neutralization with mineral acid HAn Al 2 O 3 + 6HAn ? 2AlAn 3 + 3H 2 O Extraction, filtration, drying, heating Thermal destruction of hydroxo-salts
			Al[(OH)

x An y ] 3 with (x + y) = 1 x An y ] 3 Al n+

Table 4

 4 Comparison of acetone adsorption capacity on activated carbon and hydrophobic alumina

	Activated carbon	Hydrophobic
	(coconut charcoal)	acidified alumina

Acknowledgements

The authors would like express their thanks to the Analyses Laboratory (LCA/ Axens IFP Group Technol-ogies) who contributed to the characterization of the samples.


Rochelle Ackerley
email: rochelle.ackerley@univ-amu.fr

C-tactile (CT) afferents: evidence of their function from microneurography studies in humans

C-tactile (CT) afferents are low-threshold mechanoreceptors present in the skin of humans and are thought to convey positive and pleasant aspects of touch, due to their optimal firing during gentle, caress-like contact. This review explores their role and function through the evidence produced in microneurography studies, where it is possible to record from single CTs in awake, healthy humans. CTs send a relatively-delayed signal to the brain, due to their unmyelinated, slowlyconducting axon, and are highly sensitive to small displacements of the skin, especially from dynamic, moving touch. CTs are primarily mechanoreceptors, but show some thermal sensitivity, where neutral touch (at skin temperature ~32 o C) is optimal, warm touch (~42 o C) activates them less, and cool touch (~18 o C) produces complex responses.

Highlights

• Unmyelinated C-tactile afferents can be recorded in humans via microneurography.

•

They are highly sensitive mechanoreceptors, with low activation thresholds.

•

They respond vigorously to dynamic, moving touch.

•

They show characteristic responses to mechanical, thermal, and electrical stimuli. • C-tactile afferents are believed to convey positive affective aspects of touch.

Key words: C-tactile, low threshold mechanoreceptor, microneurography, humans, affective touch, pleasant touch C-tactile (CT) afferents (Box 1) are slowly-conducting, low-threshold mechanoreceptors that respond well to gentle touch. Microneurography studies in healthy humans (Box 2) have provided a wealth of information about the physiology of CT afferents, leading to theories about their function and role in touch. Primarily, they are believed to underpin the 'affective touch hypothesis', providing positive affective input about touch, such in gentle touch between family members and for social communication [START_REF] Morrison | The skin as a social organ[END_REF][START_REF] Olausson | The neurophysiology of unmyelinated tactile afferents[END_REF][START_REF] Mcglone | Discriminative and affective touch: sensing and feeling[END_REF].

History of CT afferents

C-low threshold mechanoreceptors (C-LTMs) have been documented in animals for many years [START_REF] Zotterman | Touch, pain and tickling: an electro-physiological investigation on cutaneous sensory nerves[END_REF][START_REF] Douglas | Non-medullated fibres in the saphenous nerve which signal touch[END_REF], yet there was little evidence for the existence of their homolog in microneurography recordings in humans. This may have been due to the technical challenges of microneurography and that single C-fibers are generally more difficult to find and record from [START_REF] Vallbo | Microneurography: how it started and how it works[END_REF], thus meaning CTs were discovered later. This is also why only a handful of papers exist demonstrating CTs in humans, as there are many complex issues to consider in carrying out such investigations. In smaller nerves (e.g. the forearm, where CTs are numerous), single unit microneurography (Box 2) is more demanding, due to difficulties in accessing the nerve and stabilizing the electrode [START_REF] Vallbo | Microneurography: how it started and how it works[END_REF]. Additionally, recording from a CT is challenging, due to the small-diameter, unmyelinated Cfiber axon (~1 µm diameter, cf. ~10 m of Aβ myelinated mechanoreceptive afferents, [START_REF] Mcglone | Discriminative and affective touch: sensing and feeling[END_REF]), and their delayed response to stimulation can resemble the activity of sympathetic C-fibers efferents (Figure 1) [START_REF] Watkins | Evidence for sparse C-tactile afferent innervation of glabrous human hand skin[END_REF].

The first indication of C-LTMs in humans came in the late 1980s, where [START_REF] Johansson | Mechanoreceptor activity from the human face and oral mucosa[END_REF] found a potential C-fiber that was highly responsive to gentle touch during microneurography in the trigeminal infraorbital nerve of the face [START_REF] Johansson | Mechanoreceptor activity from the human face and oral mucosa[END_REF]. Not long after, [START_REF] Nordin | Low-threshold mechanoreceptive and nociceptive units with unmyelinated (C) fibres in the human supraorbital nerve[END_REF] showed unequivocal and thorough evidence for the existence of such touch afferents, in the trigeminal supraorbital nerve of the face, and compared their responses to C-nociceptors [START_REF] Nordin | Low-threshold mechanoreceptive and nociceptive units with unmyelinated (C) fibres in the human supraorbital nerve[END_REF]. In the following years, the co-founder of the technique of microneurography, Åke Vallbo [START_REF] Vallbo | Microneurography: how it started and how it works[END_REF] and collaborators, published a series of papers using microneurography of the antebrachial nerve of the forearm, where they postulated a role for these low-threshold C-fibers in gentle touch perception [START_REF] Vallbo | A system of unmyelinated afferents for innocuous mechanoreception in the human skin[END_REF], later naming them C-tactile (CT) afferents (Box 1) [START_REF] Vallbo | Unmyelinated afferents constitute a second system coding tactile stimuli of the human hairy skin[END_REF].

To find CTs during a microneurography experiment, the skin is touched and stroked and CTs can be identified via their delayed responses to mechanical stimulation (i.e. due to their unmyelinated axon and thus slow conduction velocity of <2 cm/s). This delayed response separates them from all other faster-conducting myelinated types of mechanoreceptive fiber (Figure 1) and confirms it is a C-fiber. To distinguish the class of C-fiber, CTs are the only afferent C-fiber type that respond vigorously to gentle touch on its receptive field (i.e. it is a low-threshold mechanoreceptor). This includes tests like having a low force activation threshold, vigorous responses to moving touch (typically at <10 cm/s), or firing well to both innocuous and noxious indentation (Figure 1). This method of characterization was initially outlined in early work on CTs [START_REF] Nordin | Low-threshold mechanoreceptive and nociceptive units with unmyelinated (C) fibres in the human supraorbital nerve[END_REF][START_REF] Vallbo | A system of unmyelinated afferents for innocuous mechanoreception in the human skin[END_REF] and was followed up in a comprehensive paper by Vallbo et al. (1999) [START_REF] Vallbo | Unmyelinated afferents constitute a second system coding tactile stimuli of the human hairy skin[END_REF], where tactile tests were delivered to the receptive field as low-force monofilament indentation (<5 mN), slow stroking, and testing blunt (innocuous) and sharp (noxious) stimuli (where CTs do not discriminate between these, but C-mechanoreceptive nociceptors show stronger responses to sharp, noxious touch). Hence, using this approach, CTs can be rapidly and unequivocally classified during microneurography, setting CTs apart from all other peripheral nerve fibers (Figure 1). A further way of distinguishing CTs from other C-fibers is the use of repetitive 2 Hz electrical stimulation of the receptive field for more than 30 s, where CTs have a characteristic response profile, showing very little slowing in their response latency (<1%; Figure 1) [START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF][START_REF] Campero | Activity-dependent slowing properties of an unmyelinated low threshold mechanoreceptor in human hairy skin[END_REF].

Physiological properties and characteristics of CTs

CTs have an average conduction velocity of ~1 m/s (between 0.3-2 m/s; [START_REF] Watkins | Evidence for sparse C-tactile afferent innervation of glabrous human hand skin[END_REF][START_REF] Johansson | Mechanoreceptor activity from the human face and oral mucosa[END_REF][START_REF] Edin | Cutaneous afferents provide information about knee joint movements in humans[END_REF][START_REF] Nordin | Low-threshold mechanoreceptive and nociceptive units with unmyelinated (C) fibres in the human supraorbital nerve[END_REF][START_REF] Vallbo | A system of unmyelinated afferents for innocuous mechanoreception in the human skin[END_REF][START_REF] Vallbo | Unmyelinated afferents constitute a second system coding tactile stimuli of the human hairy skin[END_REF][START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF][START_REF] Campero | Activity-dependent slowing properties of an unmyelinated low threshold mechanoreceptor in human hairy skin[END_REF][START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF][START_REF] **löken | Coding of pleasant touch by unmyelinated afferents in humans[END_REF][START_REF] Ackerley | Differential effects of radiant and mechanically applied thermal stimuli on human C-tactile afferent firing patterns[END_REF]), due to their small-diameter, unmyelinated axon. This relativelydelayed signal is clearly evident when the receptive field is touched and the response is recorded via microneurography. There is often a short barrage of responses from fast-conducting Aβ mechanoreceptors when the skin is stimulated, which is almost instantaneous with the touch, and a delayed response can be recorded originating from C-fibers (e.g. see figures in [START_REF] Wessberg | Receptive field properties of unmyelinated tactile afferents in the human skin[END_REF] and [START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF]). This delay can be measured by applying a mechanical tap or delivering electrical stimulation to the CT receptive field and measuring the distance between this and the microneurography recording site, to calculate the conduction velocity. Mechanical and electrical methods to establish the conduction velocity of CTs give similar values, although mechanical stimulation leads to a slightly longer delay, including time for mechanical transduction, which is by-passed by electrical stimulation [START_REF] Vallbo | Unmyelinated afferents constitute a second system coding tactile stimuli of the human hairy skin[END_REF][START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF]. Further, the unmyelinated nature of the axon is indicated by the shape of the recorded spike responses. Unmyelinated fibers typically produce a triphasic impulse, with a prominent negative deflection (Figure 1) [START_REF] Vallbo | Microneurography: how it started and how it works[END_REF][START_REF] Nordin | Low-threshold mechanoreceptive and nociceptive units with unmyelinated (C) fibres in the human supraorbital nerve[END_REF][START_REF] Vallbo | Unmyelinated afferents constitute a second system coding tactile stimuli of the human hairy skin[END_REF][START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF], which is rare in Aβ mechanoreceptive afferents [START_REF] Vallbo | Microneurography: how it started and how it works[END_REF].

Like all low-threshold mechanoreceptors, CTs have low mechanical activation thresholds, as measured by the application of calibrated monofilaments to the receptive field. CTs will respond to monofilaments of less than 5 mN (~500 mg; Table 1) and may even have thresholds as low as 0.04 mN (~4 mg) [START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF]. Some C-nociceptors respond to monofilament mechanical stimulation as low as 2.5 mN and may show a weak response to a gentle brush stroke across the

Box 1. C-tactile (CT) afferents:

A CT is defined as a mechanoreceptive afferent that is slowly-conducting, with a low mechanical threshold (<5 mN). C-tactile afferents are found in human nerves across the body, especially in hairy skin, and respond optimally to gentle, stroking touch. They have characteristic properties, such as being highly responsive to a slowly-moving stimulus and exhibiting after-discharges, and they show a specific response pattern to electrical stimulation. [START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF]. Thus, care must be taken in classifying CTs, especially compared to such C-nociceptors with lower mechanical activation thresholds. The mechanical force activation threshold and response to moving touch over the receptive field generally provides a quick and simple test to confirm a CT afferent (Figure 1). CTs will respond well to such low intensity, mechanical stimulation with a burst of spikes, whereas C-nociceptors, at most, respond with only a couple of spikes [START_REF] Vallbo | Unmyelinated afferents constitute a second system coding tactile stimuli of the human hairy skin[END_REF][START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF].

Box 2. Microneurography:

Many microneurography studies have shown that CTs respond very well to slow, moving touch, showing strong responses to a hand, brush, cotton wool, needle, or smooth metal plate stroking over the receptive field (Table 1). Early reports found that CTs do not respond particularly differently to smooth or sharp stimuli (e.g. comparing a smooth probe with a sharp needle [START_REF] Nordin | Low-threshold mechanoreceptive and nociceptive units with unmyelinated (C) fibres in the human supraorbital nerve[END_REF][START_REF] Vallbo | Unmyelinated afferents constitute a second system coding tactile stimuli of the human hairy skin[END_REF]). It is thought that response frequency is not strongly modulated by stimulus force in CTs [START_REF] **löken | Coding of pleasant touch by unmyelinated afferents in humans[END_REF], although mapping the receptive field with different indentation forces produces increases in firing frequency [START_REF] Wessberg | Receptive field properties of unmyelinated tactile afferents in the human skin[END_REF]. It is of interest to explore how CTs respond to a larger range of surfaces and forces, to give a better understanding of the role of CTs in typical every day touch interactions.

The evoked responses under controlled conditions (i.e. for force and speed) appear to be equivalent when using a soft brush [START_REF] **löken | Coding of pleasant touch by unmyelinated afferents in humans[END_REF], as compared to a smooth metal plate heated to around skin temperature [START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF], where CTs show optimal firing at velocities between 1-10 cm/s. When very slow moving touch is applied across the receptive field (<1 cm/s), CTs will fire a long barrage of spikes; however, the mean firing frequency is generally lower (~25 spikes/s) than at the optimal velocities (~40 spikes/s) [START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF][START_REF] **löken | Coding of pleasant touch by unmyelinated afferents in humans[END_REF][START_REF] Ackerley | Differential effects of radiant and mechanically applied thermal stimuli on human C-tactile afferent firing patterns[END_REF]. Conversely, when a faster moving stimulus is applied (>10 cm/s), only a handful of spikes at most are generated and these are of lower mean frequency (~25 spikes/s) than at the optimal velocities [START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF][START_REF] **löken | Coding of pleasant touch by unmyelinated afferents in humans[END_REF]. These data have been analyzed using a log10 scale for stroking velocity, where the mean firing frequency of CTs over these velocities shows a negative quadratic (inverted U-shaped) relationship. A significant quadratic curve is found in around ~90% of CT afferents and correlates with the perception of pleasantness over the same velocities [START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF][START_REF] **löken | Coding of pleasant touch by unmyelinated afferents in humans[END_REF].

Further characteristics of CTs have been documented, yet often these have only been tested in a handful of CTs. These include intermediate adaptation to a statically-applied mechanical stimulus, showing the propensity for afterdischarges, fatigue to repeated touch, and delayed acceleration of firing (after some adaptation to a static stimulus, a CT may show an increase in firing) (Table 1). It has been suggested that a potential sub-class of CT afferent may show burst firing [START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF], yet the function of this remains unknown. Very little is known about the responses of CTs to vibration, but preliminary work suggests that CTs show a comparatively poor following response to vibration, where they may follow up to 1 Hz and respond with a single phase-locked spike at vibration up to 50 Hz (Table 1). Finally, for mechanical response characteristics, CTs do respond somewhat to skin stretch, especially dynamic pulling of the skin (Table 1). Additionally, microneurographers report that CTs do not respond to gentle blowing on the skin and are not particularly sensitive to the movement of hairs [START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF], although a CT on the face located next to large scalp hairs was activated by moving the hairs [START_REF] Nordin | Low-threshold mechanoreceptive and nociceptive units with unmyelinated (C) fibres in the human supraorbital nerve[END_REF]; however, the CT response may have been mainly due to skin displacement caused by moving such large terminal hairs.

CTs are not classed as thermoreceptors, but the temperature of mechanothermal stimulation modulates their response, i.e. they are thermally-sensitive mechanoreceptors (Table 1). They show very weak, if any, responses to radiant thermal stimulation, usually only in response to cooling (~1 spike/s [START_REF] Ackerley | Differential effects of radiant and mechanically applied thermal stimuli on human C-tactile afferent firing patterns[END_REF]). Higher rates have been observed during evaporative cooling of volatile liquids from the skin (~20 spikes/s [START_REF] Nordin | Low-threshold mechanoreceptive and nociceptive units with unmyelinated (C) fibres in the human supraorbital nerve[END_REF]), which provides increased rates of skin cooling. When stationary touch is applied, a clear CT response is seen, which is lower for warmer (42 o C) touch, as compared to neutral (32 o C, skin temperature) or cool (15 o C) stationary touch [START_REF] Ackerley | Differential effects of radiant and mechanically applied thermal stimuli on human C-tactile afferent firing patterns[END_REF]. When touch is dynamic and moves across the receptive field, further differences are found. In general, neutral temperature touch (32 o C) generates responses of higher mean frequency (~40 spikes/s) than cool (18 o C) or warmer (42 o C) moving touch (~30 spikes /s) [START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF][START_REF] Ackerley | Differential effects of radiant and mechanically applied thermal stimuli on human C-tactile afferent firing patterns[END_REF]. However, at very fast velocities (30 cm/s), there is no significant difference between CT firing frequency for these three different temperatures [START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF]. This may be due to the generation of few spikes through stroking at 30 cm/s and that the thermal transfer for such a short duration of contact would be negligible.

The optimal responses of CTs to touch around skin temperature has further implicated their role in signaling affective touch between individuals, i.e. skinto-skin gentle interactions [START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF]. However, there are many more aspects that need to be considered concerning how CTs encode touch. For example, at slow stroking velocities (e.g. 0.1 cm/s), a more complex pattern is seen for mechanocool stimulation: the firing frequency is generally lower for cool touch than neutral temperature touch, yet the number of spikes generated is equivalent [START_REF] Ackerley | Differential effects of radiant and mechanically applied thermal stimuli on human C-tactile afferent firing patterns[END_REF]. This effect is found due to afterdischarges, following mechanical stimulation of the receptive field, which are more pronounced after cooling. It has been postulated that small mechanical skin contractions induced during Such afterdischarges are of low frequency and the participant does not overtly perceive any related sensation [START_REF] Ackerley | Differential effects of radiant and mechanically applied thermal stimuli on human C-tactile afferent firing patterns[END_REF], yet they may still signal some part of the cold-touch stimulus. This may potentially be a negative affective component about skin discomfort, as seen in psychophysical ratings of cold touch [START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF].

CTs can also be activated electrically, by inserting electrodes into the receptive field and by-passing the mechanical encoding mechanism. Typically, other Cfibers show pronounced increases in response latency when electrical stimulation is delivered repetitively, but this effect is negligible in CTs (Table 1). For example, it is possible to 'mark' a C-fiber (slow down its electrical latency response) using concurrent mechanical stimulation between low frequency repetitive electrical stimulation. In CTs, a small increase in latency of only 0.05% per spike elicited via the mechanical stimulation is found, compared to a 0.5% shift for C-mechanosensitive nociceptors [START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF]. Additionally, for repetitive electrical stimulation of the receptive field at 2 Hz, CT response latencies show <1% increase, which is in stark contrast to much greater response latency increases in other C-fibers [START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF][START_REF] Campero | Activity-dependent slowing properties of an unmyelinated low threshold mechanoreceptor in human hairy skin[END_REF][START_REF] Serra | Activity-dependent slowing of conduction differentiates functional subtypes of C fibres innervating human skin[END_REF]. Finally, CTs are able to follow repeated short bursts of electrical stimulation up to 20 Hz, with no latency slowing. Further, CTs can follow electrical stimulation at rates of 100 Hz, but there is some slowing of their responses; however, they do not follow electrical stimulation at 200 Hz [START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF]. Electrical stimulation also sometimes elicits additional CT firing, akin to afterdischarges [START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF][START_REF] Ackerley | Differential effects of radiant and mechanically applied thermal stimuli on human C-tactile afferent firing patterns[END_REF]. These can be modulated thermally and decrease during radiant skin warming, as compared to cooling or with no thermal stimulation [START_REF] Ackerley | Differential effects of radiant and mechanically applied thermal stimuli on human C-tactile afferent firing patterns[END_REF].

Little research has been conducted on the activation of CTs by applying chemicals to their receptive fields. Preliminary evidence suggests that CTs show negligible or low sensitivity to topical application of capsaicin (2% solution) [START_REF] Fernström | Physiological properties of unmyelinated lowthreshold tactile (CT) afferents in the human hairy skin[END_REF].

Anatomical properties and characteristics of CTs

CTs were first demonstrated on the face (infraorbital nerve [START_REF] Johansson | Mechanoreceptor activity from the human face and oral mucosa[END_REF] and supraorbital nerve [START_REF] Nordin | Low-threshold mechanoreceptive and nociceptive units with unmyelinated (C) fibres in the human supraorbital nerve[END_REF]), then many proceeding studies have focused on the hairy skin of the arm, recording from the lateral and dorsal branches of the antebrachial nerve [START_REF] Watkins | Evidence for sparse C-tactile afferent innervation of glabrous human hand skin[END_REF][START_REF] Vallbo | A system of unmyelinated afferents for innocuous mechanoreception in the human skin[END_REF][START_REF] Vallbo | Unmyelinated afferents constitute a second system coding tactile stimuli of the human hairy skin[END_REF][START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF][START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF][START_REF] **löken | Coding of pleasant touch by unmyelinated afferents in humans[END_REF][START_REF] Ackerley | Differential effects of radiant and mechanically applied thermal stimuli on human C-tactile afferent firing patterns[END_REF][START_REF] Wessberg | Receptive field properties of unmyelinated tactile afferents in the human skin[END_REF], where CTs are readily found (consisting ~40% of mechanoreceptive units sampled [START_REF] Vallbo | A system of unmyelinated afferents for innocuous mechanoreception in the human skin[END_REF]). Studies have shown the presence of CTs in the leg, including in the lateral cutaneous femoral nerve of the thigh [START_REF] Edin | Cutaneous afferents provide information about knee joint movements in humans[END_REF] and the peroneal nerve of the lower leg [START_REF] Campero | Activity-dependent slowing properties of an unmyelinated low threshold mechanoreceptor in human hairy skin[END_REF][START_REF] Löken | Unmyelinated tactile (CT) afferents are present in the human peroneal and radial nerves[END_REF], yet it seems more difficult to record CTs during leg experiments (~10% incidence at the level of the thigh [START_REF] Edin | Cutaneous afferents provide information about knee joint movements in humans[END_REF]); however, this could be somewhat biased or under-represented (e.g. by the search procedure to find afferents, by sympathetic activity masking the delayed responses).

There is evidence to suggest that CTs are readily found distally on the arm, as many are found near the hand in antebrachial experiments [START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF][START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF][START_REF] **löken | Coding of pleasant touch by unmyelinated afferents in humans[END_REF][START_REF] Ackerley | Differential effects of radiant and mechanically applied thermal stimuli on human C-tactile afferent firing patterns[END_REF], and CTs have been demonstrated both in the superficial branch of the radial nerve [START_REF] Watkins | Evidence for sparse C-tactile afferent innervation of glabrous human hand skin[END_REF][START_REF] Löken | Unmyelinated tactile (CT) afferents are present in the human peroneal and radial nerves[END_REF] and the median nerve [START_REF] Watkins | Evidence for sparse C-tactile afferent innervation of glabrous human hand skin[END_REF]. Concerning the finding of CTs in non-hairy, glabrous skin [START_REF] Watkins | Evidence for sparse C-tactile afferent innervation of glabrous human hand skin[END_REF], the preliminary evidence shows the sparse existence of mechanoreceptors that are classified using the conventional criteria as CTs, although they are seldom encountered, which suggests a far lower density, even when overall nerve mechanoreceptor density is taken into account [START_REF] Watkins | Evidence for sparse C-tactile afferent innervation of glabrous human hand skin[END_REF]. Thus, the implications of CT innervation of distal hairy and glabrous hand skin are yet to be elucidated in relation to the affective touch hypothesis and the putative glabrous skin CTs should be studied further to see whether they form a CT sub-group, although the animal literature on glabrous C-LTMs currently suggests no functional differences [START_REF] Djouhri | Electrophysiological evidence for the existence of a rare population of C-fiber low threshold mechanoreceptive (C-LTM) neurons in glabrous skin of the rat hindpaw[END_REF].

Concerning CT receptive fields, studies have shown small, spot-like zones, with one or multiple small areas of sensitivity [START_REF] Nordin | Low-threshold mechanoreceptive and nociceptive units with unmyelinated (C) fibres in the human supraorbital nerve[END_REF][START_REF] Wessberg | Receptive field properties of unmyelinated tactile afferents in the human skin[END_REF]. An in-depth study by [START_REF] Wessberg | Receptive field properties of unmyelinated tactile afferents in the human skin[END_REF] provided detailed information about CT receptive fields structure, where CTs have 1-9 small spots of sensitivity (mean = 4), with a typical area of ~10 mm 2 [START_REF] Wessberg | Receptive field properties of unmyelinated tactile afferents in the human skin[END_REF]. During electrical stimulation experiments, the latency of CT responses sometimes jumps between discrete latencies, which may be attributed to the peripheral branching of a CT axon [START_REF] Watkins | Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing[END_REF][START_REF] Campero | Activity-dependent slowing properties of an unmyelinated low threshold mechanoreceptor in human hairy skin[END_REF], thus reveals further information about the branching of the axon. It would be of interest to explore this branching further, especially if adaptation happens to certain branches and not others, and its impact on CT firing.

Conclusions

CTs are highly sensitive mechanoreceptors and it is evident that they play a clear role in encoding gentle, dynamic touch signals and conveying them to the brain. These signals are not believed to be useful for discriminative touch, due to their delayed nature of their input, but are implicated in the affective and emotional aspects of touch, such as in the reinforcement of gentle contact. There are few studies that have investigated the properties of human C-tactile afferents and further studies should be conducted into their functional characteristics (e.g. responses to different surfaces and forces, sensitivity to chemicals). Not all CTs display all the documented properties (e.g. delayed acceleration), therefore, it is of interest to explore potential anatomical and physiological sub-classes. As well as new investigations, the existing data from CTs could be analyzed further to explore how the exact firing patterns contribute to tactile perception, where other aspects (e.g. temporal components, bursting, duration of firing, differences in minimum/maximum firing rates) could reveal more about their role. This is especially complex when looking at very slow stroking touch that elicits plenty of CT activity, at highly variable rates, but where overall mean firing frequency and pleasantness is low. In a follow-up study to [START_REF] **löken | Coding of pleasant touch by unmyelinated afferents in humans[END_REF], this work showed that C-tactile afferents were tuned to both the velocity and temperature of a stroke across their receptive field. Neutral temperature moving touch (akin to skin temperature) produced higher mean instantaneous firing frequency than cooler or warmer touch.

Figure 1 .

 1 Figure 1. How to classify a C-tactile afferent in microneurography. Using two questions, it is possible to classify a C-tactile (CT) afferent, by excluding other fiber types via their specific responses. The spikes shown on the left are from a single CT, demonstrating the unmyelinated axon triphasic impulse response, with a prominent negative deflection (40 spikes overlaid, horizontal scale bar = 1 ms, vertical scale bar = 20 µV). Spikes shown on the right are from a single slowly-adapting type I (SA-I) mechanoreceptive afferent, demonstrating the difference in the myelinated axon spike shape, i.e. primarily positive-going (40 spikes overlaid, same scale bars).

  List of annotated references: papers of special interest (*) or outstanding interest (**): **Vallbo Å, Olausson H, Wessberg J: Unmyelinated afferents constitute a second system coding tactile stimuli of the human hairy skin. J Neurophysiol 1999, 81:2753-2763. This study explores and documents many different properties of C-tactile afferents, including many of their response characteristics to different mechanical stimulation. **Löken LS, Wessberg J, Morrison I, McGlone F, Olausson H: Coding of pleasant touch by unmyelinated afferents in humans. Nat Neurosci 2009, 12:547-8.This elegant study shows that C-tactile afferents show a tuning to the speed of touch to their receptive field. The mean instantaneous firing frequency was optimal at velocities of 1-10 cm/s and reduced for slower and faster velocities. This finding correlated with the perception of pleasantness of the same moving touch. **Ackerley R, Backlund Wasling H, Liljencrantz J, Olausson H, Johnson RD, Wessberg J: Human C-tactile afferents are tuned to the temperature of a skinstroking caress. J Neurosci 2014, 34:2879-83.

*

  Ackerley R, Wiklund Fernström K, Backlund Wasling H, Watkins RH, Johnson RD, Vallbo Å, Wessberg J: Differential effects of radiant and mechanically applied thermal stimuli on human C-tactile afferent firing patterns. J Neurophysiol 2018, 120:1885-1892.This study shows the complexity of CT afferent firing to different thermal and mechanical manipulations. It demonstrates that CTs respond to cooling, moreso than warming, yet the firing is typically of low frequency.

*Watkins RH, Wessberg J, Wasling HB, Dunham JP, Olausson H, Johnson RD,

Ackerley R: Optimal delineation of single C-tactile and C-nociceptive afferents in humans using latency slowing. J Neurophysiol 2017, 117:1608-1614. This work characterizes the response properties of CTs to electrical stimulation of their receptive field. It shows the unequivocal delineation between CT afferents and C-nociceptors, demonstrating that CTs form a distinct subpopulation of C-fibers, with specific characteristics.

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].

Type of stimulus Property Characteristics Papers

Mechanical

Low mechanical activation threshold A CT will have a monofilament activation threshold of <5 mN. Observed in all CTs. [7,8,18 ,9-16] Sensitive to moving touch across the receptive field CTs will fire well to a touch stimulus moved across the receptive field. This may be a hand, brush, or any other surface. Observed in all CTs.

[7,8,17 ,9-16] Responds well to both a blunt and sharp probe CTs will respond equally well to blunt or sharp indentation (compared to C-mechanosensitive nociceptors that respond well only to sharp indentation). [START_REF] Nordin | Low-threshold mechanoreceptive and nociceptive units with unmyelinated (C) fibres in the human supraorbital nerve[END_REF][START_REF] Vallbo | Unmyelinated afferents constitute a second system coding tactile stimuli of the human hairy skin[END_REF] Optimal firing for stroking velocities of 1-10 cm/s CTs show the highest mean instantaneous firing frequency for 'intermediate velocities' (1-10 cm/s), where slower and faster stroking produces lower firing frequencies. [START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF][START_REF] **löken | Coding of pleasant touch by unmyelinated afferents in humans[END_REF] Intermediate adaptation When a mechanical stimulus is applied statically to a receptive field, the CT will fire initially with a burst of spikes, then will generally adapt to the stimulus within a few seconds (cease firing). [START_REF] Johansson | Mechanoreceptor activity from the human face and oral mucosa[END_REF][START_REF] Vallbo | Unmyelinated afferents constitute a second system coding tactile stimuli of the human hairy skin[END_REF] After-discharge (firing after the removal of a stimulus) When a mechanical stimulus is removed from the CT receptive field, there will often be a response of a few spikes just after removal. [8,9,12 ,16] Fatigue to repeated stimulation

The propensity to fire (e.g. number of spikes generated, instantaneous firing frequency) will decrease somewhat when the receptive field is stimulated repetitively.

[9,11,1 7,19] Delayed acceleration (biphasic response to sustained contact) When a mechanical stimulus is applied statically to a receptive field, after the initial spike burst and intermediate adaptation, the firing may resume and even build up to a considerable rate. [START_REF] Vallbo | Unmyelinated afferents constitute a second system coding tactile stimuli of the human hairy skin[END_REF] Vibration

CTs show a comparatively poor response to vibration, where they may follow vibration to 1 Hz (few spikes) and may respond transiently with a single phase-locked spike at vibrations between 16-50 Hz.

[