Christian Montanari

Elodie Giorla

Christelle Yann Pelloux

Montanari Christian

Giorla Elodie

Pelloux Yann#

Baunez Christelle
email: christelle.baunez@univ-amu.fr

Christelle Baunez

Subthalamic nucleus mediates the modulation on cocaine self-administration induced by ultrasonic vocalizations playback in rats Running Head: STN and USV on cocaine intake

Keywords: addiction, basal ganglia, conditioned place aversion, conditioned place preference, social influence, sucrose

come

Subthalamic nucleus mediates the modulation on cocaine self-administration induced by ultrasonic vocalization playback in rats

Introduction

Drug use disorders are a major worldwide public health problem in our society (APA 2013).

Recently it was demonstrated that the so-called "proximal social factors", that are the social factors present at the time of drug exposure such as the presence and behavior of peers, play an important role in both human drug consumption and in rats self-administering drugs (Strickland & Smith 2013 for review; [START_REF] Giorla | Peer presence and familiarity as key factors to reduce cocaine intake: evidence from translational research[END_REF]. Epidemiologic studies have shown that people associated with peers who use drugs have higher probabilities to use drugs than people not associated with drug users (Bahr, Hoffmann, & Yang 2005). Moreover in a semi naturalistic bar setting [START_REF] Larsen | An experimental study on imitation of alcohol consumption in same-sex dyads[END_REF] and in a real bar study [START_REF] Larsen | The strong effect of other people's drinking: two experimental observational studies in a real bar[END_REF] it was shown that subjects in company of a heavydrinking peer consumed more alcohol than subjects in company of a light-drinking peer or an abstinent peer. In the rat, the peer presence influences drug intake in a different manner if the peer is abstinent or is also taking drugs (see Strickland & Smith 2013 and Discussion). Abuse of food, particularly of highly palatable sweet food rich in sugars, is another serious burden of our western countries (for reviews see [START_REF] Ahmed | Sugar addiction: pushing the drug-sugar analogy to the limit[END_REF][START_REF] Ahmed | Sugar addiction: pushing the drug-sugar analogy to the limit[END_REF][START_REF] Dinicolantonio | Sugar addiction: is it real? A narrative review[END_REF][START_REF] Dinicolantonio | Sugar addiction: is it real? A narrative review[END_REF]. Although there is no explicit definition of "proximal social factors" in eating-related behaviors, it is well-known that presence and behavior of peers influence also our eating behavior, a daily activity often shared with others. The probability to become obese is higher if a friend, a sibling or a spouse has become obese [START_REF] Christakis | The spread of obesity in a large social network over 32 years[END_REF]. Moreover, in presence of a noneating observer, people eat less than when alone [START_REF] Roth | Self-presentational conflict in social eating situations: A normative perspective[END_REF], and when people eat together their intake differs from when alone and depends on the others intake [START_REF] Herman | The social facilitation of eating[END_REF]. The social facilitation phenomenon of eating behavior is well-known in different animal species such as rats [START_REF] Harlow | Social facilitation of feeding in the albino rat[END_REF]), chickens (Tolman 1965), dogs [START_REF] Ross | Social facilitation of feeding behavior in dogs: II. Feeding after satiation[END_REF]) and monkeys [START_REF] Harlow | Social facilitation of feeding in the monkey and its relation to attitudes of ascendance and submission[END_REF]. [START_REF] Gardner | Imitational and social facilitatory aspects of observational learning in the laboratory rat[END_REF] showed that a rat increases its lever-pressing behavior for standard laboratory food in presence of a peer having access to food. No further studies evaluated the effect of the presence and behavior of a peer on food self-administration (SA), especially palatable sweet was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint (which this version posted October 23, 2018. ; https://doi.org/10.1101/450437 doi: bioRxiv preprint food that, like drugs, can induce a loss of control over consumption in both animals and humans [START_REF] Ahmed | Sugar addiction: pushing the drug-sugar analogy to the limit[END_REF][START_REF] Dinicolantonio | Sugar addiction: is it real? A narrative review[END_REF]. Given that the influence of proximal social factors in animals has been only recently studied, a few questions remain to be addressed, such as their influence on both drug and food SA and the ways of communication used between peers to influence each other.

Adult rats use 50-and 22-kHz ultrasonic vocalizations (USV) to respectively communicate to peers their positive and negative affective internal states (Brudzynski 2013). This acoustic communication could thus play an important role in the effects mediated by the peer presence on drug and food intake. Although recording USV of rats self-administering drugs and palatable food as sucrose is common to assess the emotional status of the animals (Browning et al. 2011;Barker, Simmons, & West 2015), no study has ever evaluated the effects of USV playback on drug and food intake, albeit other recent studies have shown that USV playback in rats is an essential tool to investigate different human brain disorders, especially those involving social communication deficits (Wöhr & Schwarting 2017). Moreover there are no studies regarding the neurobiological basis of the proximal social factor's effects on drug and food consumption. However, one can hypothesize that emotional and motivational processes may interact in the influence of proximal social factors on goal-directed behaviors. Therefore, the subthalamic nucleus (STN), a cerebral structure belonging to the basal ganglia, could represent an interesting candidate because it was previously shown to modulate affective responses to emotional stimuli [START_REF] Pelloux | The subthalamic nucleus keeps you high on emotion: behavioral consequences of its inactivation[END_REF]) and its inactivation reduces motivation for cocaine while increasing that for food only in a Progressive Ratio schedule of reinforcement but not in standard SA conditions (Baunez et al 2005;[START_REF] Rouaud | Reducing the desire for cocaine with subthalamic nucleus deep brain stimulation[END_REF]. In this study we thus aimed at evaluating the effects of 50-and 22-kHz USV playback on both cocaine and sucrose SA, and in both sham-control and STN-lesioned rats.

was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Material and methods

Animals and Surgery

A total of 37 male Lister Hooded rats (Charles River Laboratories, Saint-Germain-sur-l'Arbresle, France) weighing ~290 g at their arrival were used. Animals were housed in pairs, maintained in animal facility and subjected to intra-jugular implantation of catheters and/or STN ibotenic excitotoxic lesions (rats underwent oral sucrose SA were only subjected to STN lesions) as previously described (Baunez et al. 2005;[START_REF] Pelloux | The subthalamic nucleus keeps you high on emotion: behavioral consequences of its inactivation[END_REF]; see Supporting Information). All the rats had free access to standard laboratory food pellets (Scientific Animal Food and Engineering, Augy, France) and water, except during the experimental sessions. All animal procedures were approved by local ethic committee and the University of Aix-Marseille (#3129.01), and were in accordance with the European Community Regulations for Animal Use in Research (CEE N° 86/609) and the French regulation (Decree 2010-118).

Experiments overview

In total, 4 different experiments were performed: 1) evaluation of the effects of 50-and 22-kHz USV playback on cocaine SA; 2) evaluation of the effects of 50-and 22-kHz USV playback on sucrose SA; 3) assessment of the rewarding properties of the 50-kHz USV using the Conditioned Place Preference (CPP) paradigm; 4) assessment of the aversive properties of the 22-kHz USV using the Conditioned Place Aversion (CPA) paradigm. To limit the number of animals, the rats that first underwent the cocaine SA procedure were also tested in the CPA paradigm, and the rats tested in the CPP paradigm were subsequently used for the sucrose SA experiment (except 3 shamcontrol rats used only for the CPP paradigm). Between the two experimental procedures all animals were maintained for 2-3 months in standard housing conditions, without any exposure to cocaine, sucrose or USV playback. The rats were randomly allocated into the different experimental groups when the study was designed with the aim to use the smallest number of animals sufficient to was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

consider the results reliable (cocaine SA/CPA: sham-control rats, N=6; STN-lesioned rats, N=9; CPP: sham-control rats, N=11; STN-lesioned rats, N=9; sucrose SA: sham-control rats, N=8; STN-lesioned rats, N=9).

Playback display of 50-and 22-kHz USV over cocaine and sucrose SA sessions Apparatus: four standard SA chambers (Camden Inst, 24-cm length, 25-cm width, and 26-cm height) placed in sound-and light attenuating cubicles were used. Each chamber was equipped with two retractable levers positioned on the right-hand wall, two white stimulus lights (one above each lever), an extractable dipper for the delivery of liquids in the food magazine (placed between the 2 levers), and a white house light located in the ceiling. Acoustic stimuli were presented through ultrasonic loudspeakers (Ultrasonic Dynamic Speaker Vifa, Avisoft Bioacoustics, Glienicke, Germany) placed in an opening in the middle of the chambers ceiling (see Supporting Information).

Procedures: testing began 10 days after the surgery. Different animals were used to test the USV playback on cocaine and sucrose SA. Daily 1h sessions took place during the dark phase, between 09:00 and 17:00 hours. Immediately before the start of sessions, catheters of rats that were selfadministering cocaine were connected -through infusion lines and liquid swivels -to cocaine syringes positioned on motorized pumps (Razel Scientific Instruments, St. Albans, VT, USA) placed outside of the chambers. At the start of each session a white house light was turned on for the duration of the session and two lever were extended. Depending on the experiment, pressing the active lever (counterbalanced between rats) delivered either an intravenous infusion of cocaine (250µg/90µl/5s) or a 10% solution of sucrose accessible in the dipper (300µl/dipper/5s) and switched on the white cue light above the active lever during the reward delivery; pressing the inactive lever was recorded but had no consequence. A 20-s time-out period was imposed (and maintained during all the phases of the experiment), during which any further lever press was recorded as perseverative response but had no consequence. Few sessions were run under a continuous schedule of reinforcement (FR1; every lever press on the active lever was reinforced).

was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint (which this version posted October 23, 2018. ; https://doi.org/10.1101/450437 doi: bioRxiv preprint Then progressively over days, the number of lever presses required to obtain a reward was increased to FR2 and FR5 (the 2 nd and the 5 th lever press was respectively reinforced). When rats reached a stable cocaine or sucrose SA (baseline condition: ≤20% of variability in the number of rewards for 5 consecutive days), they were exposed -for 3 different blocks of 5 consecutive days and in the following order -to the playback of 50-/22-kHz USV, background noise, and 22-/50-kHz USV, counterbalancing between rats the starting order of the USV (50-versus 22-kHz). During the playback sessions, the first lever press of each FR5 ratio resulted in the playback of the appropriate file (1.5 sec length) and the lever pressing counting was not interrupted during the playback (see Acoustic Stimuli section and Supporting Information for further details on the USV used and the playback procedure). The background noise was used as control stimulus of possible unspecific effects not due to the communicative function of the USV. The chambers, pumps and loudspeakers were controlled by a home-built interface and by a home-written PC-program (built and written by Y. Pelloux).

Assessment of USV emotional properties through the Conditioned Place Preference/Aversion (CPP/CPA) paradigm

The rewarding and aversive properties of the USV were assessed using the CPP/CPA paradigm (for a review see Tzschentke 2007).

Apparatus: As previously described [START_REF] Rouaud | Reducing the desire for cocaine with subthalamic nucleus deep brain stimulation[END_REF]), a gray Perspex box (70 cm length x 30 cm width x 40 cm height) divided in two main lateral compartments (30 x 30 x 40 cm each) with a small intermediate chamber (10 cm in length) was used for the experiments. The two lateral compartments differed only in the spatial organization of two vertical columns (i.e. conditioned stimulus). Acoustic stimuli were played back through the same ultrasonic loudspeakers used for the SA sessions, placed above the middle of the lateral compartments of the apparatus. A camera (PNP-90/TL, Swann Communications Ltd, Central Milton Keynes, UK) connected to a Computer Video was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint (which this version posted October 23, 2018. ; https://doi.org/10.1101/450437 doi: bioRxiv preprint Interface (EzCAP USB Video Grabber, EZCAP.TV, Newton Stewart, UK) was mounted above the apparatus for video recording.

Procedures: different animals were used for the CPP and the CPA experiment. Rats that underwent the CPP experiment were exposed to the 50-kHz USV, while the ones that underwent the CPA experiment were exposed to the 22-kHz USV. Briefly, on the preconditioning day, rats explored the whole apparatus for 15 min and the time (in sec) spent in each compartment was measured via the video recording. From the following day and for 8 consecutive days (conditioning phase: 30 min/session/day), animals were confined -on alternate days-one day in one of the two lateral compartments and exposed to the USV (50-or 22-kHz USV), and the day after in the other lateral compartment and exposed to the background noise [counterbalancing between rats the lateral compartment paired with the USV and the starting order of the acoustic stimuli (USV vs background noise)]. The same acoustic files (1.5 sec length) played back during the SA sessions were displayed during each conditioning session, every 5 minutes for a total of 5 playbacks/session (see Acoustic Stimuli section and Supporting Information for further details on the USV used and the playback procedure). The day after the end of the conditioning, on the testing day, rats were again let free to explore the apparatus for 15 min without any playback and the time spent in each compartment was measured via the video recording, as in the pre-conditioning day. The rewarding or aversive properties of the USV were inferred by the measurement of the time spent during the test in the compartment previously paired with the USV in comparison with the time spent in the same compartment in the preconditioning day.

Acoustic Stimuli

Animals were tested for their auditory capacity before the beginning of the experiments, after recovery from surgery; all rats showed an excellent startle response after a sudden sound stimulation and then no rat was excluded from the experiments due to hearing deficits. The USV displayed during the experiments were recorded from 2 adult male Lister Hooded rats, unfamiliar to the tested rats and not included in any experimental procedure. A long (1.5s) 22-kHz USV call was recorded from a rat that received an electric foot shock (0.5 mA for 2s) in a SA chamber before the recording. For playback a 50-kHz USV, the length had to be identical to the 22-kHz USV. Since such long 50-kHz USV do not exist, a series of 50-kHz calls (total file length: 1.5s; total calling time: 0.45s) was recorded from a rat during the exploration of a SA chamber containing food, water and scents from its cage mate after depriving him overnight from all of the above reported stimuli (see Supporting Information for further details on the USV used).

A Condenser Ultrasound Microphone (CM16/CMPA-48, Avisoft Bioacoustics) and its Computer Audio Interface (QUAD-CAPTURE USB 2.0 Audio Capture, Roland Corporation, Los Angeles, CA, U.S) were used to record the USV with a sampling rate of 192 kHz in 16-bit format using Avisoft RECORDER (Version 4.2, Avisoft Bioacustics).

Histology

See Supporting Information

Statistical analysis

The statistical analyses have been performed using SPSS 20 software and all variables were expressed as mean number ± SEM.

Playback display of 50-and 22-kHz USV over cocaine and sucrose SA sessions: in both shamcontrol and STN-lesioned rats the number of rewards reached during the sessions was analyzed via a two-way repeated measures ANOVA with playback condition (4 levels: baseline, background noise, 50-and 22-kHz USV) and session (5 levels: five days for each playback condition) as within-subject factors. The average of the baseline condition was also compared with the number of rewards obtained during the 1 st day of the other playback conditions, through a one-way repeated measure ANOVA. Significant ANOVA effects (p ≤0.05) were followed by post hoc Fisher's LSD tests. No statistical methods were used to predetermine sample size, but the samples are comparable was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint (which this version posted October 23, 2018. ; https://doi.org/10.1101/450437 doi: bioRxiv preprint 10 to those reported in previous studies. However, power analysis (1-group, 2-tails and equal variance model) was performed, using the Free Online Power and Sample Size Calculator available at powerandsamplesize.com, to confirm that our sample sizes were sufficient to detect reliable changes in cocaine intake under USV playback influence. Comparisons between groups on the number of cocaine/sucrose rewards reached by rats during the 1 st day of playback and for the average of each 5-day block, were made using independent t-test (2-tails).

Assessment of the USV emotional properties through the Conditioned Place Preference/Aversion

(CPP/CPA) paradigm: we calculated the preference/avoidance score as the difference between the time (in sec) spent on the testing day in the compartment previously associated with the USV and the time spent in the same compartment on the preconditioning day. A positive score indicates a preference, while a negative score an avoidance, for the compartment previously paired with the USV. The conditioning effect was evaluated within each group via the non-parametric Wilcoxon signed-rank test for paired samples, while differences between groups were assessed through the non-parametric Mann-Whitney U test for independent samples.

Results

Histology

Five rats (2 and 3 from the cocaine SA/CPA and sucrose SA/CPP experiments respectively) were excluded from the STN-lesioned groups results and statistical analyses due to lesions either unilateral, too restricted or outside the STN. The sites and extent of lesions were characterized by a neuronal loss and associated gliosis (see Fig. 1 for a representative intact and lesioned STN).

Effects of 50-and 22-kHz USV playback on cocaine SA sessions

Thirteen rats were included in the analysis (sham-control group: N=6; STN-lesioned group: n=7).

was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint (which this version posted October 23, 2018. ; https://doi.org/10.1101/450437 doi: bioRxiv preprint 11 Sham-control rats: the analysis of the effects of the USV playback across all sessions revealed a significant effect of playback condition [ANOVA: F(3,15)=6.711, p=0.020], but not of session [ANOVA: F(4, 20)=3.365, p=0.060] nor playback condition × session interaction [ANOVA: F(12,60)=2.269; p=0.118]. Post hoc comparisons between the different playback conditions, illustrated in Fig. 2a and in Fig. 2b as averaged by condition, indicated that the playback of background noise did not affect cocaine intake compared with baseline (p=0.240), whereas the playback of the 50-kHz USV induced a significant decrease in the number of cocaine injections compared to baseline (p=0.048, power=0.99), background noise (p=0.034, power=0.99) and 22-kHz USV (p=0.028, power=1), and the 22-kHz USV had no significant effect when compared to baseline and background noise (p=0.244; p=0.094 respectively). Interestingly, when the analysis only included the 1 st day of each playback condition, as illustrated in Fig 2c, there was a significant playback effect [ANOVA: F(3,15)=9.802, p=0.001] and post-hoc comparisons revealed that baseline and background noise did not affect cocaine intake differently (p=0.723), and that both 50and 22-kHz USV had a significant effect, the first decreasing cocaine intake (p=0.046 with power=0.60 and p=0.041 with power=0.71, from baseline and background respectively), while the second increased it (p=0.034 with power=0.92 and p=0.024 with power=0.94, from baseline and background respectively), leading to a significant difference between 50-and 22 kHz USV (p=0.019, power=1).

STN-lesioned rats: in this group the number of cocaine injections was not affected by the different playback conditions (Fig. 2d-f). As shown in Fig. 2d was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Comparisons between groups showed that sham-control rats, in comparison to STN-lesioned rats, took a higher number of cocaine injections during the 1 st day of the 22-kHz USV playback [t(11)=2.322; p=0.42], and a lower number of injections during the 50-kHz 5-day block [t(11)=-2.837; p=0.021].

Effects of 50-and 22-kHz USV playback on sucrose SA sessions

Fourteen rats were included in the analysis (sham-control group: N=8; STN-lesioned group: N=6).

Sham-control rats: the number of sucrose rewards obtained was stable regardless of playback condition and session (Fig. 3a-c). The analysis of the number of sucrose rewards earned across all sessions did not indicate any significant effect of playback condition STN-lesioned rats: the number of sucrose rewards in STN-lesioned rats was not modulated by the different acoustic stimuli played back (Fig. 3d-f). As shown in Fig. 3d

Assessment of the rewarding and aversive properties of the 50-and 22-kHz USV

For the CPP and CPA experiments, 17 sham-control (N=11 and 6 respectively) and 13 STNlesioned rats (N=6 and 7 respectively) were included in the analysis. As shown in Fig. 4, only in the sham-control group, playback of the 50-and 22-kHz USV respectively induced a preference and an was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

avoidance towards the environment previously paired with the USV. As shown in Fig. 4a, the playback of the 50-kHz USV induced a conditioning effect for the compartment paired with the USV in sham-control but not in STN-lesioned rats (Wilcoxon signed-rank test: p=0.026 and p=0.400, respectively), leading to a significant difference between the two groups (Mann-Whitney U test: p=0.027). Similarly, and as shown in Fig. 4b, the playback of the 22-kHz USV induced a conditioning effect for the compartment paired with the call in sham-control but not in STNlesioned rats (Wilcoxon signed-rank test: p=0.028 and p=1.000, respectively), leading again to a significant difference between the two groups (Mann-Whitney U test: p=0.022).

Discussion

Affective meaning of USV and consequences on cocaine intake

We have shown here that in sham-control rats the playback of 50-kHz USV induced a decreased intake of cocaine, while the playback of 22-kHz USV increased it transiently. The 50-kHz calls may have acted as an alternative reward able to reduce cocaine intake, as it was previously reported for other rewards. It was shown indeed that four regular food pellets placed in the self-administration chambers reduced heroin intake in rats fed ad libitum [START_REF] Lenoir | Supply of a nondrug substitute reduces escalated heroin consumption[END_REF]. Moreover in a mutually exclusive choice procedure, majority of rats fed ad libitum choose to self-administer sugar solutions over cocaine and other drugs (see Ahmed, Guillem, & Vandaele 2013 for a review). The presence of a peer has also been shown to be rewarding for rats, given that they develop a social interaction-induced CPP (Calcagnetti & Schechter 1992;[START_REF] Giorla | Peer presence and familiarity as key factors to reduce cocaine intake: evidence from translational research[END_REF]) and learn to press a bar for a physical contact with another rat or only its view (Angermeier 1960). Interestingly, in the SA studies that evaluated the effects of peers on drug intake, rats tested with a non-self-administrating peer took less cocaine than rats tested with a peer also taking cocaine or rats tested alone [START_REF] Smith | Peer influences on drug self-administration: social facilitation and social inhibition of cocaine intake in male rats[END_REF][START_REF] Robinson | The effects of social contact on cocaine intake under extended-access conditions in male rats[END_REF][START_REF] Giorla | Peer presence and familiarity as key factors to reduce cocaine intake: evidence from translational research[END_REF]). Here we have shown that the playback of 50-kHz calls induced a CPP, confirming the positive meaning of the 50-kHz USV (Brudzynski 2013). This is particularly in line with the highest positive affective states conveyed by the frequency-was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint (which this version posted October 23, 2018. ; https://doi.org/10.1101/450437 doi: bioRxiv preprint modulated (FM) and trills calls (Burgdord et al. 2008;[START_REF] Burgdorf | Frequency modulated 50 kHz ultrasonic vocalizations reflect a positive emotional state in the rat: neural substrates and therapeutic implications[END_REF], of which our file is in majority composed (see Acoustic Stimuli section and Supporting Information for further details on the USV used). Our CPP result is also in agreement with the fact that rats can selfadminister playback of 50-kHz USV (Burgdorf et al. 2008), and that 50-kHz USV playback induces dopamine release (Willuhn et al. 2014) and neuronal activation [START_REF] Sadananda | Playback of 22-kHz and 50-kHz ultrasonic vocalizations induces differential c-fos expression in rat brain[END_REF] in the nucleus accumbens, these latter results being interpreted as a stimulation of the reward system. If we then consider the apparently rewarding properties of the 50-kHz USV and the ability of other rewards to reduce cocaine intake, it is not surprising that also the 50-kHz calls were able to do so. It was also well documented that 50-kHz USV serve an important affiliative pro-social function (Wöhr et al. 2016;Wöhr & Schwarting 2017). Consequently it could be possible that rats decreased their cocaine intake because they were approaching the loudspeaker and looking for a peer. However, given that in a previous report rats showed an approach behavior towards the source of the 50-kHz USV playback only during the first USV exposure (Wöhr & Schwarting 2012), we should not expect it to last as long as 5 consecutive SA sessions. Moreover, our data obtained with the CPP (a classical and well established paradigm to study the rewarding properties of drug and non-drug stimuli; see Tzschentke 2007 for a review), showed that rats, during the test day, kept approaching the compartment associated with the USV in absence of their playback. Considering the 50-kHz USV are uttered during -mostly positive -various social interactions (Burgdorf et al. 2008), but also after exposure to food and drugs (Browning et al. 2011;Barker, Simmons, & West 2015), or in mildly aversive situations as a short social isolation (Wöhr et al. 2007), we can reasonably suppose that the social and rewarding aspects of the 50-kHz USV could overlap in some circumstances, while in other situations one aspect could prevail on the other. Further research is necessary to investigate this point.

In another hand, the playback of the 22-kHz USV probably increased cocaine intake through the induction of an aversive state, in line with a series of results indicating higher levels of cocaine was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint (which this version posted October 23, 2018. ; https://doi.org/10.1101/450437 doi: bioRxiv preprint intake after exposure to stressful stimuli in rats [START_REF] Goeders | Non-contingent electric footshock facilitates the acquisition of intravenous cocaine self-administration in rats[END_REF]. In support of this hypothesis, the long 22-kHz call used here induced a CPA, confirming the aversive meaning of the 22-kHz USV, in particular of the long ones (Brudzynski et al. 1993;Brudzynski 2013; see Acoustic Stimuli section and Supporting Information for further details on the USV used). Moreover previous findings indicated that rats avoid SA of 22-kHz USV playback (Burgdorf et al. 2008), and that their playback induces neuronal activation in areas correlated to fear regulation such as amygdala and periaqueductal grey area [START_REF] Sadananda | Playback of 22-kHz and 50-kHz ultrasonic vocalizations induces differential c-fos expression in rat brain[END_REF]. Interestingly, starting from the 2nd day of playback, the 22-kHz USV had no effect on cocaine intake, suggesting that rats could have habituated to the aversive properties of the calls [START_REF] Rankin | Habituation revisited: an updated and revised description of the behavioral characteristics of habituation[END_REF]). However none was observed with the 50-kHz USV, and repeated 22-kHz USV playback was able to inducein the same animals -a CPA after 2-3 months from the end of the cocaine SA experiment, ruling out the hypothesis of an habituation process. Alternatively, the 22-kHz USV could be less negative than 50-kHz USV were rewarding, albeit in both cases we used highly affective stimuli (see above and Supporting Information). Taken all together, our results seem to suggest that the aversive properties of the 22-kHz USV were apparently maintained along the SA sessions, without however interfere with cocaine consumption, possibly because animals have quickly understood the absence of any physical threat.

The USV-induced modulation is specific to cocaine

Our results also indicate that the USV ability to affect cocaine intake in sham-control rats is specific to the drug-related behavior, as it was not observed when animals had access to sucrose, suggesting that the rewarding properties of sucrose overcame the emotive properties of the USV. Interestingly, although the rats were fed ad libitum, the number of sucrose rewards was much higher than the number of cocaine infusions (122.91±5.95 sucrose dippers versus 8.57±0.95 cocaine infusions).

Even if it can be argued that the rewarding value of a sucrose reward could be lower than a cocaine infusion, there is a large literature showing that sugar can be addictive, inducing binge-like behavior was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint (which this version posted October 23, 2018. ; https://doi.org/10.1101/450437 doi: bioRxiv preprint and withdrawal symptoms in rodents (DiNicolantonio, O'Keef, & Wilson 2017), and that addictive properties of sugar can be indeed stronger that those of cocaine and other drugs such as nicotine and heroin [START_REF] Lenoir | Intense sweetness surpasses cocaine reward[END_REF]Vandaele et al. 2016;[START_REF] Huynh | Rats quit nicotine for a sweet reward following an extensive history of nicotine use[END_REF]. This has been recently explained by the "delay drug reward hypothesis" [START_REF] Ahmed | Trying to make sense of rodents' drug choice behavior[END_REF], suggesting that the delay between an intravenous drug injection and its actions on the brain reward systems diminishes "apparently" its rewarding efficacy, as opposed to the immediate rewarding effect of sugar. In another hand it could be argued that the sucrose experiment was conducted 3 months after the rats were exposed to 50-kHz USV in the CPP experiment, exposure that may prevent the playback of USV to affect sucrose SA. However, if a former exposure to USV was sufficient to prevent them to modulate behavior, the exposure to 22-kHz USV in the cocaine SA experiment should have prevented the development of a CPA induced by 22-kHz USV playback tested 3 months later. Since CPA was observed, we can rule this hypothesis out.

The neurobiological substrate for such modulation requires intact STN

The last noteworthy finding of our research is the inability of the 50-and 22-kHz USV playback to affect both cocaine and sucrose SA in STN-lesioned rats. It could be due to the fact that STNlesioned rats were not able to decode the emotional properties of the USV. In fact STN-lesioned rats did not develop a preference or an aversion for the CPP/CPA compartment respectively paired with the 50-and 22-kHz USV, in agreement with their blunted emotional responses to both positive and negative stimuli previously reported [START_REF] Pelloux | The subthalamic nucleus keeps you high on emotion: behavioral consequences of its inactivation[END_REF]. The STN has long been considered a motor structure and is the target of the surgical treatment for patients with Parkinson's disease using the High Frequency Stimulation (HFS), also called Deep Brain Stimulation (DBS) [START_REF] Limousin | Effect of parkinsonian signs and symptoms of bilateral subthalamic nucleus stimulation[END_REF]. Although the action mechanisms of the DBS are still not fully understood, they seem to partly mimic the effects of lesions of the targeted area [START_REF] Gubellini | Deep brain stimulation in neurological diseases and experimental models: from molecule to complex behavior[END_REF]. Interestingly STN DBS of Parkinsonian patients alters the processing of emotive information regardless the positive or negative stimulus valence, and regardless the visual or auditory sensory modality (for a review see was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint (which this version posted October 23, 2018. ; https://doi.org/10.1101/450437 doi: bioRxiv preprint [START_REF] Péron | Subthalamic nucleus: a key structure for emotional component synchronization in humans[END_REF]. Regarding the acoustic sensory modality, Parkinson's disease patients under STN DBS show deficits in identifying different vocal emotions [START_REF] Péron | Recognition of emotional prosody is altered after subthalamic nucleus deep brain stimulation in Parkinson's disease[END_REF] and in contrast, decoding of emotional vocal expressions in healthy subjects involves an activation of the STN in functional magnetic resonance imaging (fMRI) studies (Péron et al. 2016). Taken all together, these human discoveries and our findings in STN-lesioned rats suggest that STN is a crucial node through which the emotional context modulates motivational processes, but also highlight possible sideeffects in PD patients that should be monitored carefully. These results are also relevant for other pathologies for which STN can be a surgical target, such as Obsessive Compulsive Disorders (OCD; [START_REF] Mallet | Subthalamic Nucleus Stimulation in Severe Obsessive-Compulsive Disorder[END_REF] or cocaine addiction [START_REF] Pelloux | Deep brain stimulation for addiction: why the subthalamic nucleus should be favored[END_REF]. Indeed, highlighting the possible risk to impair emotions in patients treated with STN DBS should lead to a better monitoring of these patients. In another hand, this decrease in emotional functioning might explain the beneficial effects of STN DBS in OCD patients by possibly reducing a pathological overemotionality.

Conclusions

In conclusion we found that, in sham-control rats, 50-and 22-khZ USV are respectively rewarding and aversive. Playback of the rewarding USV leads to decreased cocaine intake, while playback of the aversive one increased it transiently. Interestingly these effects are specific to the drug-related behaviors as they were not observed when animals had access to sucrose. The neurobiological substrate for these behavioral observations seems to involve the STN since STN-lesioned rats were not able to decode the USV emotional content, as expressed by the lack of any effect in both the CPP/CPA and the cocaine SA experiments. These results demonstrate how social acoustic communication and positive contexts created either by USV or presence of a peer are important in rats to diminish drug intake and how STN modulation could help preventing detrimental effect of negative emotional and motivational contexts. These results can translate to the influence of proximal social factors on drug intake described in human drug users [START_REF] Giorla | Peer presence and familiarity as key factors to reduce cocaine intake: evidence from translational research[END_REF] was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint (which this version posted October 23, 2018. ; https://doi.org/10.1101/450437 doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint (which this version posted October 23, 2018. ; https://doi.org/10.1101/450437 doi: bioRxiv preprint

  and in Fig. 2e as averaged by condition, the analysis across the sessions did not indicate a significant effect of playback condition [ANOVA: F(3,18)=0.800, p=0.468], session [ANOVA: F(4,24)=0.371, p=0.772] or playback condition x session interaction [ANOVA: F(12,72)=0.600, p=0.624]. As shown in Fig. 2f, no playback condition effect was further found on the 1 st day of playback [ANOVA: F(3,18)=0.653, p=0.513].

  [ANOVA: F(3,21)=0.898, p=0.433], session [ANOVA: F(4,28)=1.315, p=0.289] or playback condition x session interaction [F(12,84)=0.898, p=0.530], as shown in Fig. 3a and in Fig. 3b as averaged by condition. As shown in Fig. 3c, no significant playback condition effect was further found in the analysis of the 1 st day of playback [ANOVA: F(3,21)=1.376, p=0.284].

  and in Fig. 3e as averaged by condition, no significant effect of playback condition [ANOVA: F(3,15)=1.802, p=0.190], session [ANOVA: F(4,20)=1.065, p=0.383] or playback condition x session interaction [ANOVA: F(12,60)=0.913, p=0.458] was found. As illustrated in Fig. 3f, the absence of a significant playback condition effect was confirmed in the analysis of the first 1 st day of playback [ANOVA: F(3,15)=1.246, p=0.328].

Figure 1 .

 1 Figure 1. Frontal STN sections stained with cresyl violet

Figure 2 .

 2 Figure 2. Effects of 50-kHz USV, 22-kHz USV and background noise playback on the mean

Figure 3 .

 3 Figure 3. Effect of 50-kHz USV, 22-kHz USV and background noise playback on the mean

Figure 4 .

 4 Figure 4. Conditioned Place Preference and Conditioned Place Aversion for the compartment

Figure 2

 2 Figure 2

  and couldwas not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. risk reduction strategies. They are also critical for all patients that could be subjected to STN lesions or DBS for PD, OCD or even addiction, who should be monitored carefully regarding their emotional state. Supporting Information is available online at Addict Biol's website. Angermeier WF (1960) Some basic aspects of social reinforcements in albino rats. Journal of Smith MA (2013) The effects of social contact on drug use: behavioral mechanisms controlling drug intake. Exp Clin Psychopharmacol 22(1):23-34. DOI: 10.1037/a0034669. Tolman CW (1965) Emotional behaviour and social facilitation of feeding in domestic chicks. . Acoustic Communication in Rats: Effects of Social Experiences on Ultrasonic Vocalizations as Socio-affective Signals. Curr Top Behav Neurosci 30:67-89. DOI: 10.1007/7854_2015_410.

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	thus help improving Comparative and http://dx.doi.org/10.1037/h0047435. Accessed 13 Sept 2018. Physiological Psychology 53 Bahr SJ, Hoffmann JP, Yang X (2005) Parental and peer influences on the risk of adolescent drug (4): 364-367. Available at: Strickland JC, Anim Behav 13(4):493-6. PMID: 5882808. Wöhr M, Engelhardt KA, Seffer D, Sungur AÖ, Schwarting RK (2017)
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Acknowledgements

This research was funded by CNRS, Aix-Marseille Université (AMU), the "Agence Nationale pour la Recherche" (ANR_2010-NEUR-005-01 in the framework of the ERA-Net NEURON to CB and supporting YP), the Fondation pour la Recherche Médicale (FRM DPA20140629789 to CB), and the support of the A*MIDEX project (ANR-11-IDEX-0001-02) funded by the « Investissements d'Avenir » French Government program, managed by the French National Research Agency (ANR). All the authors declare no competing interests.

Author contribution

CM designed and performed the research, analyzed data and wrote the paper; EG performed experiments; YP designed the research and contributed to analytic tools; CB designed and supervised the research, performed surgery and wrote the paper.


Laëtitia Mayen

Nicholai D Jensen

Maximilien Desbord

Danielle Laurencin

Christel Gervais

Christian Bonhomme

Mark A Smith

Florence Porcher

Erik Elkaim

Cédric Charvillat

Mark E Smith

Pierre Gras

Christian Rey

Jérémy Soulié

Christèle Combes
email: christele.combes@ensiacet.fr

Advances in the synthesis and structure of α-canaphite: a multitool and multiscale study

Keywords: Canaphite, Calcium pyrophosphates, Synthesis, Synchrotron X-ray and neutron diffraction, Structure refinement, DFT, NMR, dehydration

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

Introduction

Condensed phosphate structures have received much less attention than orthophosphate ones. All polyphosphates are indeed hydrolyzed in aqueous solutions, and it has long been considered that this hydrolytic degradation was the cause of the quasi-absence of polyphosphate geological minerals.

Pyrophosphates (i.e. diphosphates P 2 O 7 4-) are the most stable polyphosphate anions, and interestingly, hydrated calcium pyrophosphate salts are encountered in both geological and biological environments. 1- 3 In the field of geological minerals, the calcium disodium pyrophosphate tetrahydrate, also called canaphite (CaNa 2 P 2 O 7 .4H 2 O), has been identified as the first example of condensed phosphate geological mineral. 2 The only forms of persistent polyphosphates identified in a biological extracellular medium are the calcium pyrophosphate dihydrates Ca 2 P 2 O 7 .2H 2 O (monoclinic and triclinic, m-CPPD and t-CPPD respectively), which occur in joints of patients suffering from pseudogout. [START_REF] Khon | [END_REF][5] Pritzker et al. 6 also reported the possible formation of canaphite in vitro after 24 h at 10°C or 37°C in a crystallization model system involving sodium pyrophosphate and calcium chloride diffusion in cadaveric articular hyaline cartilage (raw or denaturated). Moreover, Mandel et al. 7 studied the in vitro modeling of m-CPPD and t-CPPD crystal deposition, and their results revealed the formation of α and β-canaphite crystals as minor phases in a biological grade gelatin gel at physiological pH.

Few studies related to canaphite have been published so far. Two polymorphs of canaphite, namely α and β have been identified as monoclinic structures and the γ polymorph structure remains unknown. 2,8 Cheng et al. 9 obtained the β-canaphite phase by diffusion of solutions in hydrogel-like media mimicking the synovial fluids, and they were able to determine its crystal structure. Subsequently, Cheng and Pritzker 8 synthesized the α-canaphite phase at room temperature by adding a sodium pyrophosphate solution at neutral pH into a calcium salt solution. They showed that this α phase crystallized in the monoclinic system with Pc space group, but the positions of hydrogen atoms were not determined. Later, Rouse et al. 2 confirmed this monoclinic structure and reported extensive interatomic distances and angles data, making a comparison to the data from Cheng and Pritzker. 8 The hydrogen atom positions were still missing. Following these initial studies, Cave [START_REF] Cave | Investigation of layered calcium phosphates and related materials for biomaterial applications[END_REF] synthesized α-canaphite according to a modified protocol from Cheng and Pritzker. 8 This involved pouring together cationic and anionic solutions whereupon precipitation occurred without the need of neutralizing the pyrophosphate solution.The precipitate was then matured overnight. Examination of the XRD pattern presented suggested that an amorphous phase might be present in the as-synthesized α-canaphite sample. This observation is supported by the estimated number of structural water molecules from thermogravimetric analysis (3.5 per formula unit) as this is not consistent with the stoichiometry of pure α-canaphite. Another method claimed α-canaphite could be prepared by mechanical grinding of a mixture of precursor salts for several hours in the presence of water, leading to a fairly pure phase, containing 5% w/w of impurities. [START_REF] Safronova | [END_REF] α and β canaphite both possess a layered structure which could confer specific dehydration and ion exchange properties to these compounds. The sodium ions exchangeability in canaphite was successfully investigated by reflux reaction of canaphite for two hours in potassium nitrate saturated solution leading to anhydrous potassium pyrophosphate phase following a structural rearrangement. [START_REF] Cave | Investigation of layered calcium phosphates and related materials for biomaterial applications[END_REF] A FTIR and Raman vibrational spectroscopic study of synthetic α-canaphite did not present evidence of sample purity. [START_REF] Hubert Joe | [END_REF] To the best of our knowledge, no solid state NMR studies of canaphite polymorphs have been reported so far, in contrast to other crystalline calcium pyrophosphate phases, which have been studied by 1 H, 31 P and 43 Ca magic angle spinning (MAS) solid state NMR techniques. [13][14][15][16] A small number of papers concerning canaphite to date have all referred to the synthesis protocol by Cheng and Pritzker 8 , with some adaptations. However, both the protocol and its modifications are poorly described in the literature. In addition, the synthetic canaphite samples were not fully characterized to check for their purity before further structural investigations.

Considering the potential development of canaphite-based biomaterials and/or canaphite identification/diagnosis in the biomedical field, it is essential to thoroughly study its structure. Thus will allow better understanding of disease associated with calcium pyrophosphate-based crystal deposition and the related material properties. In addition it will allow the potential of such a compound as an innovative bioactive bone substitute material based on enzyme-and/or pH-driven pyrophosphate

(P 2 O 7 4-
) hydrolysis into orthophosphate (PO 4

3-

) to be better understood. 17 In this context the present study initially described a robust protocol for the synthesis of pure α-canaphite. Then an advanced multiscale characterization methodology combining synchrotron and laboratory experimental techniques and computational modeling tools is applied to thoroughly characterize the phase from the atomic to the macroscopic scales. This provides a full solution of crystal structure, including the positions of hydrogen atoms.

Materials and Methods

Precursors

Calcium chloride dihydrate salt (CaCl 2 .2H 2 O, 100.2%, VWR Chemicals) was used as received. The pyrophosphate precursor, anhydrous tetrasodium pyrophosphate (Na 4 P 2 O 7 ), was prepared by heating disodium hydrogen phosphate (Na 2 HPO 4 , 99%, Merck) at 400°C during 15 hours in a muffle furnace.

The purity of the as-prepared sodium pyrophosphate precursor was checked by XRD, Raman and 31 P solid state NMR spectroscopies (no residual orthophosphate was detected, Figure S1) before its use for α-canaphite synthesis.

α-canaphite synthesis α-canaphite powder was synthesized by double decomposition in solution (precipitation) at room temperature using an original protocol. Calcium and phosphate reagent solutions were prepared separately by dissolving 2.11 g of CaCl 2 •2H 2 O (0.0144 mole) in 80 mL of deionized water, and 17.71 g of the as-prepared sodium pyrophosphate (Na 4 P 2 O 7 ) precursor (0.0666 mole) in 800 mL of deionized water. The calcium solution was then added to the pyrophosphate solution at a constant volumetric flowrate (32 mL.min -1 ) using a peristaltic pump. After complete addition, the suspension was left to mature for 24 hours under magnetic stirring, and then filtrated, and washed with 800 mL of deionized water. The precipitate was finally dried at 37°C during one night and then stored at -20°C to prevent for any structural evolution before characterization. This synthesis has been performed at least in triplicate to check for its reproducibility.

In view of the fine structural study by neutron diffraction and the FTIR and Raman band assignments, deuterated canaphite was prepared following the protocol detailed above, but using deuterated water (D 2 O, Sigma Aldrich, Deuterium Oxide 99 atom% D) instead of deionized water to prepare both precursor solutions and as washing medium after filtration. In addition, several precautions were taken at each step of the synthesis protocol to prevent as much as possible any deuterium-hydrogen exchange with water vapor present in the air during powder transfers and storage (hermetically sealed flasks, drying by lyophilization, storage first in a dessicator with a deuterated water saturated atmosphere).

Finally, the sample was vacuum-packed in a plastic bag and stored in the freezer before its analysis.

α-canaphite characterizations

The as-synthesized α-canaphite powder was fully characterized using complementary multiscale laboratory and synchrotron and neutron techniques for structural, microstructural, molecular and elemental investigations, in combination with computational DFT modeling tools.

Powder X-ray diffraction (XRD).

A preliminary XRD analysis was performed at room temperature using a laboratory X-ray diffractometer (INEL Symphonix 1000) with a cobalt anticathode (λ(K α ) = 1.788970 Å) and a curved counter to rapidly check the sample purity. Then the as-synthesized α-canaphite powder was further analyzed on a synchrotron facility to collect data for structure refinement. Synchrotron powder X-ray diffraction patterns of the synthesized α-canaphite were acquired at 21°C on the twocircle diffractometer of the CRISTAL beamline at the SOLEIL synchrotron (Gif-sur-Yvette, France) as part of the project No. 20130932 "Structural investigations on hydrated calcium pyrophosphate phases of biological interest: study on synthetic and biological samples". A monochromatic beam was selected, using a Si (111) double-crystal monochromator, and its wavelength (0.5815 Å) was determined using NIST standard LaB 6 . The powder sample was placed in a 0.7 mm-diameter special glass capillary mounted on a spinner to improve averaging. High angular resolution was obtained with the 21 perfect Si (111) crystal rear analyzer mounted on a two-circle diffractometer. Detection was performed using Scionix YAP: Ce scintillation detectors.

Neutron diffraction analysis. In order to locate precisely the D atoms, neutron powder diffraction (NPD) measurements were performed on the synthesized deuterated α-canaphite. The experiment was carried out at the G44 diffractometer on the S2 cold source at the Orphée-LLB reactor (France). The diffractogram was recorded at 233 K using a Ge004 monochromator at a neutron wavelength of 1.959 Å between 2θ = 6.00° and 161.30° with steps of 0.05°. Between 149.00° < 2θ < 152.00° the cryofurnace used gave rise to a parasitic peak and this zone had to be excluded from subsequent analyses.

Structure refinements. D atoms were located from difference Fourier maps calculated with the NPD data. Subsequent Rietveld refinements were performed using the JANA 2006 software from joint NPD and synchrotron XRD data. Because this procedure imposed a common value of the cell parameters for the two set of measurements (synchrotron X-ray and neutron diffraction measurements), both wavelengths were refined in order to absorb potential discrepancies in peaks positions; the refined values remained close to the initial values. Distance (H-O distances within 0.001 Å), angle restraints (H-O-H = 104.45(1)°) and equivalence between all isotropic thermal displacement parameters were imposed for the refinement of the water molecules. All the representations of the refined structure presented in this paper were obtained using Vesta. 18 FTIR and Raman vibrational spectroscopic analysis. A Raman Labram HR 800 confocal microscope Horiba Yvon Jobin and a laser radiation provided by a 532 nm Argon diode laser with a power of 14 mW were used. Each spectrum was acquired with an integration time of 60 seconds and three accumulations. FTIR spectra were obtained using a Nicolet IS50 FT-IR spectrophotometer (Thermo Scientific) at 4 cm -1 resolution and 64 scan accumulation, by transmission from a KBr pellet. α-canaphite and it deuterated analogue were both analyzed by FTIR and Raman spectroscopy.

Scanning Electron Microscopy (SEM).

SEM observations were performed on a LEO 435 VP microscope. Samples were stuck on double-faced carbon tape and silver sputter-coated before observation.

Thermal analyses. Thermogravimetric (TGA) and differential thermal analysis (DTA) were performed in triplicate using a Setaram instrument (Setsys Evolution System) from 20°C to 1000°C with a step of 30 min at 1000°C, and a heating rate of 4°C per minute in air flow.

Inductively Coupled Plasma -Optical Emission Spectrometry (ICP-OES).

The analyses were performed on an ICP Ultima Expert machine (argon flow: 15 L/min) to determine the calcium, phosphorus and sodium concentrations in triplicate.

Ultra-High Pressure phase Liquid Chromatography (UHPLC). A Waters Acquity apparatus coupled

with an evaporative light scattering detector (ELSD) was used for the detection and titration of traces of chloride ions. Measurements were done in triplicate.

Solid state NMR.

The experiments were performed on the non-deuterated α-canaphite samples to analyze the local environments of 23 Na, 31 P, 43 Ca and 1 H nuclei. All solid state NMR spectra were recorded at 14.1 T on a VNMRS-600 MHz spectrometer, except for 1 H NMR experiments, which were recorded at 20.0 T on a Bruker 850 MHz NMR spectrometer (NEO 4 console). A 1 H spin-echo NMR spectrum was acquired using a 1.3 mm HXY MAS probe tuned to 1 H (ν 0 = 850.23 MHz). The experiment was performed at 40 kHz MAS. The spectra were recorded using a 2.1 μs 90° and 4.2 μs 180° 1 H pulse, with 1 rotor period echo delay, and using 2 s recycle delay. A total of 16 scans were collected. The experiments were performed with the temperature control unit set to -10 °C. 1 H chemical shifts were referenced to the methyl groups in alanine (δ iso ( 1 H) = 1.20 ppm). A 31 P single pulse experiment was performed using a 3.2 mm HXY MAS probe tuned to 1 H (ν 0 = 599.82 MHz) and 31 P (ν 0 = 242.81 MHz), and spinning at 18 kHz. A 3 μs 90° excitation pulse was used (RF ≈ 83 kHz), and spinal-64 1 H decoupling (RF = 100 kHz) during acquisition. A recycle delay of 800 s was used for full relaxation, and 24 scans were collected. 31 P chemical shifts were referenced to the phosphate group in synthetic hydroxyapatite, Ca 10 (PO 4 ) 6 (OH) 2 (δ iso ( 31 P) = 2.8 ppm). A 23 Na single pulse MAS NMR spectrum was recorded using a 3.2 mm HXY MAS probe spinning at 20 kHz, and the temperature control unit set to 0 °C. The experiment was performed using a 0.5 μs 30° 23 Na solid pulse (RF ≈ 83 kHz) and spinal-64 1 H decoupling (RF ≈ 100 kHz) during acquisition. A 1 s recycle delay was used, and 6000 scans were collected. The 23 Na NMR chemical shifts were referenced to a 1.0 M NaCl aqueous solution (δ iso ( 23 Na) = 0 ppm). A 43 Ca MAS NMR spectrum was recorded using a 9.5 mm probe, spinning at 4 kHz. A 3 μs 43 Ca excitation pulse was used (30° solid pulse, RF ≈ 15 kHz), preceded by a DFS pulse of 4 ms, with a DFS sweep from 250 kHz to 60 kHz in order to increase sensitivity. The spectrum was recorded using a 5 s recycle delay and 31700 scans were co-added. The chemical shifts were referenced to a 1 M CaCl 2 aqueous solution (δ iso ( 43 Ca) = 0 ppm), while powers were calibrated on a saturated CaCl 2 solution. No temperature control was applied.

Computational study. The unit cell parameters were set to the combined neutron/synchrotron X-ray diffraction data and kept fixed during geometry optimizations to ensure consistency between experimental and optimized structures. Atomic positions were then relaxed with the VASP (Vienna Abinitio Simulation Package) code 19 based on the Kohn-Sham Density Functional Theory (DFT) and using a plane-wave pseudopotential approach. Two situations were tested: relaxing H positions only, or all atomic positions. The NMR parameters were then calculated within Kohn-Sham DFT using the QUANTUM-ESPRESSO code [20][21] , keeping the atomic positions equal to the values previously calculated with VASP. The PBE generalized gradient approximation 22 was used and the valence electrons were described by norm conserving pseudopotentials 23 in the Kleinman Bylander form. 24 The shielding tensor was computed using the Gauge Including Projector Augmented Wave (GIPAW) approach 25 , which enables the reproduction of the results of a fully converged all electron calculation 26 .

The isotropic chemical shift δ iso is defined as δ iso = -[σ -σ ref ] where σ is the isotropic shielding and σ ref is the isotropic shielding of the same nucleus in a reference system. σ ref was fixed so that the average sum of experimental and calculated shifts of a series of reference compounds coincide, typically calcium phosphates and pyrophosphates for 31 P and 43 Ca, 27 and sodium phosphates and silicates for 23 Na. The principal components V xx , V yy , and V zz of the electric field gradient (EFG) tensor defined as |V zz | ≥ |V xx | ≥ |V yy | are obtained by diagonalization of the calculated tensor. The quadrupolar interaction can then be characterized by the quadrupolar coupling constant C Q and the asymmetry parameter η Q , which are defined as: C Q = eQV zz /h and η Q = (V yy -V xx )/V zz (e is the proton charge, h Planck's constant and Q the quadrupole moment of the considered nucleus). Q values of 104 and -44.4 mb were used for 23 Na and 43 Ca respectively, in agreement with the values reported by Pyykkö 28 and Bryce. 29

Results and discussion

Composition of the synthesized powder

Preliminary X-ray diffraction laboratory analysis of the as synthesized canaphite powder showed that the sample corresponds to the α-canaphite phase according to ICSD data (n°26015) (Figure S2). The elemental composition (Ca, Na and P) of the powder was determined by ICP-OES spectrometry (Table 1). The experimental values were in agreement with the theoretical ones. In addition, attempts to detect chloride ions (counter ions from the calcium salt precursor, i.e. CaCl 2 .2H 2 O) using UHPLC were unsuccessful (below the detection limit, i.e. < 1.00 10 -6 mol of Cl -in 100 mg of sample). Such thorough elemental characterizations which were not performed in previous published studies, are essential to further study and discuss on the related structure and properties of α-canaphite. TGA analysis of the synthesized α-canaphite showed that the mass loss, related to the water release, occurred in two steps between ≈ 70°C and ≈160°C (confirmed by the first derivative curve in red, Figure 1b). The mass loss is stabilized at ≈ 400°C corresponding to the total removal of water. The total mass loss reached 21.4 ± 0.5 % and allowed determining the number of water molecules associated to the assynthesized α-canaphite: 3.92 ± 0.09. This value is close to that expected theoretically. The DTA analysis curve (Figure 1a) revealed the presence of an exothermic peak at about 440°C which corresponds to a recrystallization phenomenon. The as-synthesized α-canaphite powder was treated at 400°C and 600°C during 15 h to identify the recrystallization product. The XRD analysis of both heattreated powders (at 400°C and 600°C) revealed the formation of the same anhydrous crystalline phase:

α-CaNa 2 P 2 O 7 (triclinic) which has been already described by Bennazha et al. 30 However, a shift of the diffraction peaks leading to slightly different unit cell parameters compared to the data reported in the literature was observed (data not shown). The current refined structural data of the as-prepared α-CaNa 2 P 2 O 7 are presented in Table S1. The dehydration of α-canaphite will be discussed later, after the presentation of its fine structural characterization. Morphological characterization of the synthesized α-canaphite crystals.

SEM was used to determine the morphology of the α-canaphite particles (Figure 2). The particles presented a bundle-like morphology, including platelet-like crystals gathered from the middle of the bundle. The characteristic size of these platelets was approximately 11.0 μm in length, 2.0 μm in width and 0.1 μm in thickness (Figure 2). The platelet morphology of the individual α-canaphite crystals could be related to its layered structure. The bundle-like organization of the as-prepared α-canaphite crystals is similar to that of natural canaphite mineral referenced in mineralogy database (for example: mindat.org). It can be hypothesized that such bundle-like crystal agglomerates result from a spherulite formation process which generally occurs when the driving force (i.e. supersaturation) is quite high or when crystals habit is highly oriented; the platelet-like morphology of α-canaphite crystals could favor this organization as polycrystalline growth system. 31 Category 1 spherulites are known to grow radially from the nucleation site whereas category 2 spherulites start to grow like thread fibers leading to a crystals sheaf after branching which finally ended as a sphere after longer evolution times. The organization of α-canaphite platelet crystals observed by SEM (Figure 2) corresponds to a category 2 spherulites growth (Figures 2b and2c). However it appears that some of the observed category 2 spherulites are not perfectly symmetric and some of the platelets crystals are disassembled and/or fragmented (Figure 2a); the 24h-maturation under magnetic stirring, filtration, and washing are steps for canaphite synthesis that can have altered the growth of these spherulites. The formation of category 2 spherulites was also identified for monoclinic calcium pyrophosphate tetrahydrate (m-CPPT β) and dihydrate (m-CPPD) crystals formation from amorphous calcium pyrophosphate evolution in solution at pH = 5.8 and 50°C. 32 Fine structural characterization of the as-synthesized α-canaphite phas.

The as-synthesized non-deuterated and deuterated α-canaphite samples were analyzed by synchrotron X-ray and neutron diffraction, respectively (Figure 3). Then Rietveld refinements were performed from joint neutron and synchrotron-X-ray diffraction data (Table 2). Computer programs: Jana2006

The solved α-canaphite structure presents molecular groups that correspond to pyrophosphate ions and four water molecules, as expected. The asymmetric unit has a composition corresponding to the formula 3. The structure of the α-canaphite is composed of alternating pyrophosphate and water layers (oriented according to the plane (001)) separated by layers of calcium and sodium cations (Figure 4).

Hydrogen bonds ensure the cohesion of molecules within the layers (Figure 5). The P-O-P angle of the pyrophosphate group has often been discussed as an adaptive parameter in a number of structures. Its value can be determined from structural data (129.9(3)° and 129.8° as determined from both neutron/synchrotron X-ray diffraction data refinement and DFT calculations, respectively, Table 3) or from vibrational spectroscopic data as reported by Rulmont et al. [START_REF] Rulmont | [END_REF] and other workers 34 with an empirical equation based on the relative position of symmetric and antisymmetric stretching vibrations of the POP group. Such a determination for α-canaphite, 129°, [START_REF] Hubert Joe | [END_REF] is in agreement with that obtained from the crystal structure. Torsions between the terminal groups are low, linking the canaphite structure to dichromate-type structures which is a common type of structure for hydrated calcium pyrophosphate compounds like triclinic calcium pyrophosphate dihydrate (t-CPPD), monoclinic calcium pyrophosphate tetrahydrate (m-CPPT β), anhydrous calcium pyrophosphate and also the anhydrous calcium disodium pyrophosphate α phase (α-CaNa 2 P 2 O 7 ). 29,35,36 As mentioned by Cheng et al. 9 , the α-and β-calcium disodium pyrophosphate tetrahydrate structures are very similar, the main differences being the organization of the Ca 2+ ‧‧‧P 2 O 7 4-‧‧‧Ca 2+ chains in relation to the plan (010). Figure 7 highlights the close relationship between the two structures, including the organization of the CaNa 2 P 2 O 7 .4H 2 O units. The most significant differences can be observed on the alternating orientations of pyrophosphate molecules. The intra-and intermolecular distances (Table 3)

Synchrotron XRD Neutron diffraction

are significantly similar to those of the β-canaphite structure. 9 Complementarily, a methodology combining experimental (solid state NMR) and computational methods (first principles GIPAW calculations 25 ) was implemented to characterize in detail the structure of α-canaphite at the atomic level. The 31 P, 1 H, 23 Na, 43 Ca MAS NMR spectra and the corresponding extracted NMR parameters (isotropic chemical shifts, quadrupolar constants, C Q , and quadrupolar asymmetries, η Q ) are given in Figure 8 and Table 4, respectively.

It should be noted that calculations were performed on structural models deriving from the structure previously presented (Figures 4 to 6), in which either only H atom positions, or all atomic positions were relaxed by DFT. In the latter case, as discussed below, the agreement with experimental NMR data shows the best agreement. Therefore, P-O, Ca-O, Na-O and O-H bond lengths obtained after relaxation were added in Table 3. It appears that P tetrahedra and Ca/Na octahedra geometries are not significantly modified by relaxation. On the other hand, Ow-H bond lengths originally around 0. MAS NMR spectroscopy can act as a highly reliable tool of investigation to check the purity of a given α-canaphite powder sample. The 23 Na MAS spectrum is more complex as 23 Na is a quadrupolar nucleus (I = 3/2). Nevertheless, it can be interpreted by the superposition of two contributions (Na1, Na2, see Table 4) broadened by residual second-order quadrupolar interaction (it is well established that MAS cannot suppress entirely such kind of second order broadening). Though less direct than 31 P MAS NMR analysis, 23 Na MAS NMR remains a complementary method to check the sample purity. The 43 Ca NMR spectrum exhibits a rather featureless peak corresponding to the unique calcium atom in the asymmetric unit. The broadening of the line is mainly due to residual second order quadrupolar effect as well, because calcium-43 is a spin 7/2 quadrupolar nucleus. Finally, the 1 H spin echo MAS NMR spectrum is probably the most difficult to interpret as residual 1 H-1 H homonuclear dipolar coupling broadens the line shapes (even spinning at 40 kHz at the magic angle). The shifted 1 H resonance (above 6 ppm) is in agreement with the presence of some strong H-bonds within the canaphite structure.

Table 4: 31 P, 23 Na, 43 Ca, 1 H experimental and extracted data for the as-synthesized α-canaphite powder.

Computational details related to GIPAW calculations 25 are given in the Materials and Methods section.

Tot rel calc: GIPAW data obtained after DFT relaxation of all positions (see caption of Table 3). Rel H calc: GIPAW data obtained after DFT relaxation of H positions only. GIPAW DFT calculations of the NMR parameters were performed on structural models deriving from the neutron/synchrotron X-ray structures, in which either only H atom positions, or all atomic positions were relaxed by DFT (Table 4). These calculations were found to be in very good agreement with the experimental data, confirming the accuracy of the final structure of α-canaphite. In the case of 31 P NMR, results obtained after total relaxation of the α-canaphite atomic positions led to improved agreement with experimental shifts. More importantly, for 31 P and 23 Na, the GIPAW calculations could be used for assigning both sites resolved in NMR spectroscopy. Regarding 1 H NMR, the overall range of calculated 1 H chemical shifts was found to be in complete agreement with the corresponding experimental data shown in Figure 8. Moreover, as previously observed for other pyrophosphate phases, such GIPAW DFT calculations show the strong impact of H-bonding on the 1 H isotropic chemical shifts. 13 If the positions of the protons are not precise enough, the GIPAW calculations usually diverge.

Rel H calc Tot rel calc Experimental

δ iso / ppm C Q / MHz η Q δ iso / ppm C Q / MHz η Q δ iso / ppm C Q / MHz η Q P1 -1.
Vibrational spectroscopic analysis of the as-synthesized α-canaphite phase (deuterated and nondeuterated).

α-canaphite was characterized by FTIR and Raman spectroscopy as well. The vibrational spectrum of crystals can be interpreted using the factor group theory which predicts the number of lines expected and their activity in IR and Raman. Focusing on internal vibration modes of H 2 O(D 2 O) and P 2 O 7 4molecular units, respectively 24 and 42 vibration lines should be expected (see details in supporting information on Vibrational spectroscopies and in Table S2 and Table S3). The Raman full spectrum of the as-synthesized α-canaphite powder are presented in Figure S3 and the H 2 O(D 2 O) and P 2 O 7 4molecular vibrational domains (FTIR and Raman) in Figure 9. The different FTIR and Raman lines and their respective assignments for α-canaphite and its deuterated analogue are reported in Table S4.

Obviously all the 66 lines predicted for the internal vibration modes cannot be distinguished, due to line broadening and superimpositions.

Generally several vibrational domains can be distinguished depending of the vibration energy. For P 2 O 7 4stretching vibrations (1200-700 cm -1 ), the two PO 3 groups and the POP bridge are considered and their vibration domain is well identify (Figure 9a). In the 1200-1000 cm -1 range, the two PO 3 groups of each pyrophosphate ion should show different lines (three for each group, distinguished as one symmetric and two antisymmetric vibrations), and the group factor effect could split again these lines. Generally one very strong ν s line is visible on the Raman spectrum of α-canaphite and many other pyrophosphates salts. [START_REF] Hubert Joe | [END_REF]37 For the POP bridge two stretching modes, symmetric and antisymmetric, are found in the free ion (950-700 cm -1 ); in α-canaphite these could also be split according to factor group considerations. In the spectra obtained only one ν s POP line is seen at the same position (740-743 cm -1 ) in Raman spectrum and the one of FTIR deuterated α-canaphite, in the FTIR H 2 O canaphite spectrum this line seems hindered by the broad water libration line at this position. This line much intense in Raman than in IR, is a specific signature of pyrophosphate ions. A ν as POP line is observed in all samples (FTIR and Raman) at 906-909 cm -1 , in addition a stronger line is seen at 938-934 cm -1 only in FTIR, the attribution of which is not clear. Lower energy vibrations corresponding to bending modes of the P 2 O 7 group are also present, but they seem more difficult to assign considering the other contributions, especially those of generally broad water libration lines. For H 2 O vibrations, the four water molecules in the formula unit show different environments and should exhibit 4 lines for each ν s , ν as and δ vibration modes of the molecule (Figure 9b). These lines would also be split in virtue of group factor considerations. The Raman and FTIR lines (Figure 9) are in close positions according to the group theory treatment predicting identical irreducible representations, vibrational energy levels and transitions, for both techniques. This observation confirms the absence of inversion centre in the P 2 O 7 4-groups as already noted from structural data.

The deuteration allows to distinguish water bands from pyrophosphate ones especially in the bending domains where H 2 O libration lines can be easily shifted towards lower wavenumbers (Figure 9b). Such lines can be identified at about 800, 706, and 548 cm -1 especially. The observation of a faint line at 791 cm -1 , in the deuterated sample FTIR spectrum, is attributed to an incomplete deuteration of the samples We showed by thermogravimetric analysis that α-canaphite recrystallized at about 440°C into the anhydrous calcium disodium pyrophosphate triclinic phase (α-CaNa 2 P 2 O 7 ). The latter phase is not a layered structure (Figure S4) and the orientation of the pyrophosphate molecules are very different from that in the α-canaphite structure for which strong hydrogen bonds are also involved as demonstrated by the present fine structural analysis by diffraction methods, solid state NMR and DFT calculations. These observations suggest that this transformation (recrystallization) is probably complex with strong structural reorganization. In addition, the observed dehydration mechanisms could be related to the different coordinations of Ow4, Ow9 and Ow10 atoms, only with sodium ions, and Ow8 with sodiumcalcium binding (Figures 4 to 6). Indeed the latter related water molecule (involving Ow8) could be the last one leaving the structure due to the two cations binding. This hypothesis is supported by the fact that one water molecule was also differentiated in another hydrated calcium pyrophosphate layered compound, monoclinic calcium pyrophosphate tetrahydrate beta (m-CPPT β), and identified as the last one to leave the structure upon heating and also the one involved in pyrophosphate internal hydrolysis into orthophosphate. 34 In the case of α-canaphite structure, we can hypothesize that the Ow8-related water molecule which is also the one the closer to pyrophosphate (distance Ow8-O11 = 3.123 Å) could be the one involved in the internal hydrolysis of pyrophosphate ions. Figure S5 showed the Raman spectrum of α-canaphite dried at 250°C, an intermediary temperature, i.e. higher than 37°C but lower than that leading to α-canaphite full dehydration (440°C): we can observed the appearance of a new band of low intensity at 950 cm -1 characteristic of ν s PO 4 supporting the hypothesis of the formation of orthophosphate via a pyrophosphate internal hydrolysis. Currently we are thoroughly investigating the effect of temperature on the α-canaphite phase and especially its transformation up to the dehydrated phase (α-CaNa 2 P 2 O 7 ) involving among others an hydrolysis of pyrophosphate ions upon moderate heating which could stabilize a potential intermediary amorphous phase formation; this study will be published in a forthcoming manuscript.

Conclusion

Canaphite is a layered phosphate of biological interest from the perspectives of in vivo calcium pyrophosphate-based crystals-associated disease or as an innovative bone substitute material based on enzyme-and/or pH-driven pyrophosphate (P 2 O 7 4-) hydrolysis into orthophosphate (PO 4

3-

). The novel synthesis protocol developed here is simple, reproducible and enables pure α-canaphite, CaNa 2 P 2 O 7 .4H 2 O to be obtained. SEM allowed identifying the organization of α-canaphite platelet crystals corresponding to a category 2 spherulite development and, TGA-DTA analysis, the dehydration of α-canaphite into anhydrous calcium disodium pyrophosphate through a recrystallization process above 440°C. Various observations of the phase development are presented. Most importantly for the first time, the monoclinic crystal structure of α-canaphite was fully solved (including the positions of hydrogen atoms) by a combination of synchrotron X-ray and neutron diffraction data, solid state NMR and DFT calculations. This detailed structural analysis identifies the presence of some strong H-bonds within the α-canaphite structure. One of the four oxygens from water molecules has a different coordination i.e. the two-cation binding of Ow8. It is therefore probable that this water is the last one to leave the structure during canaphite dehydration. In addition, the Ow8-related water molecule is most probably involved in the internal hydrolysis of pyrophosphate ions than the other three water molecules as it is the closest to pyrophosphate molecule. A thorough study is now necessary of the α-canaphite dehydration process which will be fully described in a forthcoming paper. The very high signal-to-noise available from 31 P and 23 Na MAS NMR spectroscopies are suitable to confirm the purity of any sodium pyrophosphate precursor and α-canaphite. 31 P MAS NMR provides much sharper spectral lines such that both P atoms of the unique pyrophosphate group in the asymmetric unit are completely resolved.

The first principles GIPAW calculations provide unambiguous assignments both for 31 P and 23 Na resonance peaks and an interpretation for the scattering of the 1 H isotropic chemical shifts related to the H-bond network in the α-canaphite structure. Finally, vibrational spectroscopies, especially Raman is a promising tool for fast in vitro, in vivo or ex vivo calcium disodium pyrophosphate microcrystals identification/diagnosis needing only a small amount of sample. This study provides a full description of α-canaphite phase and some initial thermal property determination. Both are essential to fully exploit the structural and physico-chemical properties of α-canaphite in materials science, especially for biomedical applications.

Figure 1 .

 1 Figure 1. DTA (a) and TGA (b) curves of the synthesized α-canaphite powder.

Figure 2 :

 2 Figure 2: SEM micrographs of the synthesized α-canaphite crystals.

Figure 3 :

 3 Figure 3: Rietveld refinement plots from powder synchrotron X-ray diffraction data (a) and neutron diffraction data (b) of the synthesized α-canaphite (non-deuterated (a) and deuterated (b)). Experimental and simulated diagrams and difference are shown; vertical lines indicate the Bragg peak positions. (The grey zone around 150° on neutron pattern (b) corresponds to a parasitic peak related to the cryofurnace, which is excluded for data refinement).

CaNa 2 P 2 O 7 .

 227 4H 2 O, each atom occupying a 2a Wyckoff position of the Pn space group. The pyrophosphate molecule is thus non-centrosymmetric. Selected bond lengths and angles are reported in Table

Figure 4 .Figure 5 .

 45 Figure 4. Three-dimensional representation of the monoclinic elemental cell of α-canaphite, with placement of hydrogen atoms (obtained from refinement of both X-ray and neutron diffraction data), (yellow: sodium, blue: calcium, grey: phosphorus, red: oxygen, white: hydrogen)

Figure 6 .

 6 Figure 6. Representation of calcium and sodium polyhedra of the α-canaphite structure (yellow: sodium, blue: calcium, grey: phosphorus, red: oxygen, white: hydrogen).

Figure 7 .

 7 Figure 7. Comparison of β (a) and α (b) canaphite structures, the (010) plans of interest are shown in blue, (yellow: sodium, blue: calcium, grey: phosphorus, red: oxygen, white: hydrogen).

  83 Å increase up to almost 1 Å after relaxation, a value closer to what is usually expected in water. In parallel the H••••Ow distances decrease after relaxation since the O••••O distances are maintained. Moreover, the global architecture of H-bond network is not modified since O-H••••O angles do not change significantly.

Figure 8 .

 8 Figure 8. 31 P, 23 Na, 43 Ca MAS NMR spectra at 14.1 T and 1 H spin echo MAS NMR spectrum at 20.0 T of the as-synthesized α-canaphite powder. All experimental details are given in the Materials and Methods section.

(

  about 20% of residual H 2 O), possibly due to an exchange in air during sample preparation and analysis, Deuteration shifts the water lines to the low energy region (590, 520 and 410 cm -1 ) altering the observation of P 2 O 7 4-ones especially in FTIR. The shift ratio between H 2 O and D 2 O lines, about 1.35,is in agreement with theoretical and experimental determinations.38 The FTIR and Raman determination of the POP angle using the Harcharras' diagram37 based on Lazarev observations is close to 129° in agreement with the value obtained from diffraction data.

Figure 9 .

 9 Figure 9. FTIR and Raman spectra of the synthesized α-canaphite (deuterated and non-deuterated) showing the PO 3 and POP group vibration domain (a) and the water vibration domain (b).

Table 1 :

 1 Chemical composition (Ca, P and Na amount in mmoles/100 mg of sample and Ca/P and Ca/Na atomic ratios) of the synthesized α-canaphite powder determined by ICP-OES spectrometry compared with the expected values for pure canaphite.

		Ca	P	Na	Ca/P	Ca/Na
		mmole/100 mg	mmole/100 mg	mmole/100 mg
	Synthesized	0.298 ± 0.002	0.587 ± 0.004	0.592 ± 0.001	0.508 ± 0.006 0.503 ± 0.003
	α-canaphite
	Theoretical	0.301	0.602	0.602	0.5	0.5
	composition

Table 2 :

 2 Experimental and structure refinement details of synchrotron X-ray and neutron diffraction data.

Table 3 :

 3 Selected geometric parameters of the α-canaphite structure (Å, °). P-O, Ca-O and Na-O bond lengths (Å) (P1, P2, Ca1, Na1, Na2). Exp: data obtained from the refinement of both neutron/synchrotron X-ray diffraction data. Tot rel: data obtained after DFT relaxation of all positions, starting from Exp positions (see Materials & Methods). O-P-O (°) angles, hydrogen bond lengths (Å) and angles (°) are given as well.

		P1 tetrahedron	P2 tetrahedron
			Exp	Tot rel		Exp	Tot rel
		P1-O2	1.508(4) 1.5324 P2-O1	1.523(4) 1.5299
		P1-O5	1.542(3) 1.5424 P2-O3	1.536(4) 1.5399
		P1-O7	1.536(4) 1.5232 P2-O6	1.517(3) 1.5247
		P1-O11 1.620(4) 1.6278 P2-O11 1.631(4) 1.6572
	Ca1 octahedron		Na1 octahedron			Na2 octahedron
	Exp	Tot rel		Exp	Tot rel			Exp	Tot rel
		P1 tetrahedron			P2 tetrahedron			P1-O11-P2
		Exp	Tot rel		Exp	Tot rel Exp	Tot rel
	O2-P1-O5	111.2(2)	111.1	O1-P2-O3	111.0(2)	112.6	129.9(3)	129.8
	O2-P1-O7	112.7(2)	113.3	O1-P2-O6	115.7(2)	115.2
	O2-P1-O11	108.53(2)	108.1	O1-P2-O11	103.6(2)	101.5
	O5-P1-O7	111.24(2)	112.0	O3-P2-O6	112.7(2)	112.4
	O5-P1-O11	108.53(2)	107.9	O3-P2-O11	106.9(2)	106.8
	O7-P1-O11	104.3(2)	103.8	O6-P2-O11	106.0(2)	107.2
	D-H…A	D-H	H…A			D---A	D-H…A (°)
		Exp	Tot rel	Exp	Tot rel	Exp		Tot rel Exp	Tot rel
	Ow4-H2-Ow10	0.83(1)	0.977	2.20(2)	2.02	2.945(5)	2.912	150(2)	150.8
	Ow4-H3-O7	0.83(3)	0.994	1.86(2)	1.699	2.659(5)	2.686	162(3)	171.3
	Ow8-H5-O5	0.83(2)	0.999	1.96(1)	1.717	2.772(5)	2.712	167(3)	172.8
	Ow9-H6-O5	0.83(2)	0.982	2.11(2)	1.905	2.924(5)	2.882	166(3)	172.5
	Ow10-H8-O3	0.83(2)	0.996	2.05(3)	1.802	2.861(6)	2.786	167(3)	168.9
	Ow9-H11-O1	0.83(2)	0.991	1.91(2)	1.767	2.737(5)	2.75	175(3)	170.7
	Ow8-H13-O2	0.83(2)	0.984	2.25(2)	2.022	3.000(5)	2.969	151(3)	160.9

Acknowledgments

The authors would like to thank the Agence Nationale de la Recherche (PyVerres project -grant n°ANR-16-CE19-0013) for supporting this research work, Laboratoire Léon Brillouin and synchrotron Soleil for the provision of beamtimes (proposals n° 745 and n°20130932, respectively).

The 850 MHz solid-state NMR Facility used in this research was funded by EPSRC and BBSRC, as well as the University of Warwick including via part funding through Birmingham Science City Advanced Materials Projects 1 and 2 supported by Advantage West Midlands (AWM) and the European Regional Development Fund (ERDF).. NMR spectroscopic calculations were performed using HPC resources from GENCI-IDRIS (Grant 097535).

Author's contribution

Supporting information

Tables: S1, S2, S3, S4 Figures: S1, S2, S3, S4, S5