Keywords: melanopsin, ipRGCs, pineal gland, photoreceptor Highlights : -

A subpopulation of pineal projection neurons expresses the melanopsin gene opn4xa -This new pineal cell type shares a developmental program with other projection neurons -The pineal gland contains both opn4xa-LIGHT OFF and opn4xa+ LIGHT ON projection neurons

Summary

The zebrafish pineal organ is a photoreceptive structure containing two main neuronal populations (photoreceptors and projections neurons). Here we describe a subpopulation of projection neurons that expresses the melanopsin gene, opn4xa. This new pineal cell type, that displays characteristics of both projection neurons and photoreceptors, share a similar dependency for BMP and Notch signalling pathways with classical non-photosensitive projection neurons (PN). Functionally, however, whereas classical, opn4xa-negative PNs display an achromatic LIGHT OFF response, the novel cell type we describe exhibit a LIGHT ON character that is elicited by green and blue light. Taken together, our data suggest a previously unanticipated heterogeneity in the projection neuron population in the zebrafish pineal organ raising the question of the importance of these differences in pineal function.

Functional heterogeneity in the pineal projection neurons of zebrafish

Dora Sapède 1,2 , Clair Chaigne 1 , Patrick Blader 1 and Elise Cau 1* 1 Centre de Biologie du Développement (CBD, UMR5547), Centre de Biologie Intégrative (CBI, FR 3743), Université de Toulouse, CNRS, UPS, France 2 Present address IRMB, Université de Montpellier, INSERM, Montpellier, France.

Introduction

The zebrafish pineal gland is a neuroendocrine structure of the dorsal diencephalon that plays a key role in mediating the effects of the circadian clock on sleep/wake rhythms. An important part of this activity is controlled by pineal photoreceptors (PhRs) that secrete the sleep-promoting hormone melatonin (Ben-Moshe [START_REF] Livne | Genetically Blocking the Zebrafish Pineal Clock Affects Circadian Behavior[END_REF][START_REF] Gandhi | Melatonin is required for the circadian regulation of sleep[END_REF]. The PhR population is heterogeneous and contains three described subtypes: cells expressing red cone opsin (15 cells at 48-54 hpf), those expressing exorhodopsin (20 cells at 48-54 hpf; [START_REF] Cau | A Notch-mediated, temporal asymmetry in BMP pathway activation promotes photoreceptor subtype diversification[END_REF][START_REF] Clanton | Fgf signaling governs cell fate in the zebrafish pineal complex[END_REF] and cells coexpressing parietopsin (a green-sensitive opsin) and the UV-sensitive opsin, parapinopsin1 (6 cells at 48-54 hpf; [START_REF] Cau | A Notch-mediated, temporal asymmetry in BMP pathway activation promotes photoreceptor subtype diversification[END_REF][START_REF] Koyanagi | Diversification of non-visual photopigment parapinopsin in spectral sensitivity for diverse pineal functions[END_REF][START_REF] Wada | Color opponency with a single kind of bistable opsin in the zebrafish pineal organ[END_REF]. In addition to PhRs, the pineal gland contains projection neurons (PNs, 20 cells at 48 hpf) which project to the ventral diencephalon [START_REF] Wilson | Stereotyped pathway selection by growth cones of early epiphysial neurons in the embryonic zebrafish[END_REF] and a population of AgRP2+ cells that do not express neuronal markers nor exhibit a neuronal morphology, and share molecular characteristics with retinal-pigment epithelium cells [START_REF] Shainer | Novel hypophysiotropic AgRP2 neurons and pineal cells revealed by BAC transgenesis in zebrafish[END_REF][START_REF] Shainer | Agouti-Related Protein 2 Is a New Player in the Teleost Stress Response System[END_REF].

While a significant amount is known concerning the subpopulations of pineal PhRs and their function (Ben-Moshe [START_REF] Livne | Genetically Blocking the Zebrafish Pineal Clock Affects Circadian Behavior[END_REF][START_REF] Cau | A Notch-mediated, temporal asymmetry in BMP pathway activation promotes photoreceptor subtype diversification[END_REF][START_REF] Clanton | Fgf signaling governs cell fate in the zebrafish pineal complex[END_REF][START_REF] Gandhi | Melatonin is required for the circadian regulation of sleep[END_REF][START_REF] Koyanagi | Diversification of non-visual photopigment parapinopsin in spectral sensitivity for diverse pineal functions[END_REF][START_REF] Wada | Color opponency with a single kind of bistable opsin in the zebrafish pineal organ[END_REF], much remains to be discovered concerning a possible corresponding heterogeneity in the PN population. PN functions have not been investigated. Electrophysiological experiments in lampreys and goldfishes suggest that PNs receive and integrate inputs from the PhRs [START_REF] Meissl | Neural response mechanisms in the photoreceptive pineal organ of goldfish[END_REF][START_REF] Uchida | Signal transmission from pineal photoreceptors to luminosity-type ganglion cells in the lamprey, Lampetra japonica[END_REF]. Most projection neurons are thought to function in a LIGHT OFF fashion. Indeed, electrophysiological experiments performed in the rainbow trout suggest that teleost pineal projection neurons fire constantly in the dark and exhibit a LIGHT OFF response. This response is elicited at all visible wavelengths, although maximum firing occurs at green wavelengths. While it is not clear whether individual projection neurons (PNs) respond to all wavelengths, nevertheless, this response was referred to as 'achromatic' as it is elicited by a wide range of wavelengths [START_REF] Dodt | PHOTOSENSITIVITY OF THE PINEAL ORGAN IN THE TELEOST, SALMO IRIDEUS (GIBBONS)[END_REF][START_REF] Morita | Lead pattern of the pineal neuron of the rainbow trout (Salmo irideus) by illumination of the diencephalon[END_REF]. In contrast, in the same species, few projection neurons show a chromatic response. This response is excitatory at medium and long wavelengths and inhibitory in the violet-UV range [START_REF] Morita | Lead pattern of the pineal neuron of the rainbow trout (Salmo irideus) by illumination of the diencephalon[END_REF]. PN achromatic LIGHT OFF responses were also recorded in other species such as frog, pike and turtle [START_REF] Falcón | The Photosensory Function of the Pineal Organ of the Pike (Esox lucius L.) Correlation Between Structure and Function[END_REF][START_REF] Meissl | Extraocular Photoreception of the Pineal Gland of the Aquatic Turtle Pseudemys scripta elegans[END_REF][START_REF] Morita | Nervous activity of the frog's epiphysis cerebri in relation to illumination[END_REF] while chromatic LIGHT ON responses were described in lizards, frogs and pikes [START_REF] Dodt | Mode of action of pineal nerve fibers in frogs[END_REF][START_REF] Dodt | The pineal and parietal organs of lower vertebrates[END_REF][START_REF] Meissl | Change of threshold after light-adaptation of the chromatic response of the frog's pineal organ (Stirnorgan)[END_REF]. These results suggest heterogeneity in the population of pineal projection neurons in a number of species. However, whether a similar heterogeneity exists in zebrafish is unclear, as are the molecular mechanisms that permit PN responses to changes in brightness and wavelength.

In the main photosensitive structure of the vertebrate, the retina, there is a clear heterogeneity in the population of projection neurons. Indeed, based on morphological, molecular, electrophysiological and/or functional criteria around 30 categories of Retinal Ganglion Cells, or RGCs, have been described so far in the mouse (see Sanes and Masland, 2015 for a review). Strikingly, among these subpopulations, a subset of the RGCS has been shown to be directly photosensitive, owing to the expression of melanopsin, a blue-green photosensitive pigment. These intrinsically photosensitive RGCs (ipRGCs), integrate light information from rods and cones, as well as from their innate photosensitivity, to regulate photo-entrainment of the circadian system [START_REF] Lucas | How rod, cone, and melanopsin photoreceptors come together to enlighten the mammalian circadian clock[END_REF]. Therefore, ipRGCs form a class of atypical photoreceptors that can both directly sense light and project to distinct brain areas to influence circadian rhythms, suggesting they function both as photoreceptors and projection neurons.

In this paper, we describe that the expression of the zebrafish melanopsin gene opn4xa is restricted to a subset of pineal PNs and analyse their development. The development of PhRs and PNs require specific signalling pathways: the Bone Morphogenetic Protein (BMP) pathway is necessary and sufficient to trigger PhR fate as well as to activate the Notch pathway in these cells, which inhibits a PN fate [START_REF] Cau | Notch activity in the nervous system: to switch or not switch?[END_REF][START_REF] Cau | Notch resolves mixed neural identities in the zebrafish epiphysis[END_REF][START_REF] Quillien | BMP signaling orchestrates photoreceptor specification in the zebrafish pineal gland in collaboration with Notch[END_REF][START_REF] Sapède | The pineal gland from development to function[END_REF].

Analysis of embryos deficient for the BMP or the Notch signalling pathways suggest that Notch inhibits the production of opn4xa+ cells, as it does with the rest of the PN population, and that the BMP pathway is dispensable for opn4xa+ cell development.

These results suggest that opn4xa+ cells and classical PNs share a common basic developmental program. Finally, monitoring of the induction of the immediate early gene c-fos in different illumination regimes highlights two distinct activities in the PN population. A subpopulation of opn4xa-cells show a LIGHT OFF response when the embryos are adapted in white, blue, red or green light. In contrast, opn4xa+ PNs exhibit a LIGHT ON response following a pulse of white, blue or green light. We thus propose a parallel between opn4xa+ PNs and the ipRGCs of the retina.

Results

A subset of pineal projection neurons expresses the melanopsin gene opn4xa

To describe photoreceptor heterogeneity in the pineal, we previously analysed the expression of classical opsin photo-pigments [START_REF] Cau | A Notch-mediated, temporal asymmetry in BMP pathway activation promotes photoreceptor subtype diversification[END_REF]. We also considered the expression of the five zebrafish melanopsin genes and found a small population of opn4xa+ cells at the left and right borders of the pineal organ (Fig. 1A) [START_REF] Matos-Cruz | Unexpected diversity and photoperiod dependence of the zebrafish melanopsin system[END_REF]. Combining in situ hybridisation for opn4xa with immunostaining against GFP in the transgenic lines Tg(aanat2:GFP) y8 and Tg(elavl3:EGFP) knu3 , which label PhR and PN respectively (Fig 1B, [START_REF] Cau | Notch resolves mixed neural identities in the zebrafish epiphysis[END_REF]), revealed that opn4xa expression is not detected in classical PhR (Fig. 1C,D,E) but in PNs (Fig. 1F,G,H). opn4xa expression can be first detected at 22-23 hours postfertilisation (hpf) in 1-2 elavl3:EGFP-positive cells. At 23 hpf an average of 4 opn4xa+ cells are observed and this number remains stable to at least 10 days post-fertilisation (dpf; Fig. 1L). To further characterise opn4xa+ cells, we looked for different markers displaying similarly restricted expression within the pineal organ. We found that the gene encoding the Wnt transcriptional effector Tcf7 [START_REF] Veien | Expression pattern of zebrafish tcf7 suggests unexplored domains of Wnt/beta-catenin activity[END_REF] is expressed in a small subset of PNs (Fig S1 A-C). We took advantage of a tcf7 enhancer trap line Et(T2KHG) nns8 [START_REF] Nagayoshi | Insertional mutagenesis by the Tol2 transposon-mediated enhancer trap approach generated mutations in two developmental genes: tcf7 and synembryn-like[END_REF] to characterise tcf7 expression in the pineal further. We first confirmed that expression of GFP from the Et(T2KHG) nns8 line colocalises with endogenous tcf7 expression in PNs (Fig. S1 A-C). We next assessed colabelling of the Et(T2KHG) nns8 enhancer trap line with opn4xa mRNA at stages ranging from 24 hpf to 4 dpf. Up to 30 hpf, the majority of GFP+ cells were also opn4xa-positive (Fig. 1 I,J,K Fig. S1 G). In contrast, from 2 dpf onwards, a population of cells showing a lower level of GFP appeared in the pineal of Et(T2KHG) nns8 that do not express opn4xa (Fig. S1 D-G). Altogether, we describe a subpopulation of PNs expressing opn4xa that can be labelled specifically with the Et(T2KHG) nns8 enhancer trap line up to 30 hpf.

Specification of the opn4xa+ pineal fate does not require Wnt signalling

The fact that the tcf7 enhancer trap line Et(T2KHG) nns8 is expressed in opn4xa+ cells prompted us to investigate whether Wnt signalling was involved in the specification of the opn4xa+ fate. First, we tested the expression of opn4xa in Et(T2KHG) nns8 homozygous embryos as the enhancer trap insertion abolishes expression of the endogenous tcf7 gene [START_REF] Nagayoshi | Insertional mutagenesis by the Tol2 transposon-mediated enhancer trap approach generated mutations in two developmental genes: tcf7 and synembryn-like[END_REF]. Pineal opn4xa+ cells were observed in normal numbers in all GFP+ embryos coming from Et(T2KHG) nns8 +/-incrosses suggesting that tcf7 is not required for their specification (Fig 2 A,B) as one third of GFP+ embryos are expected to be homozygous for the insertion. To look at a possible redundancy with other Wnt effectors, we analysed the expression of tcf7l2 and lef1 in the pineal and compared it with the Et(T2KHG) nns8 enhancer trap. Only the former is expressed in the pineal but did not show co-expression with the tcf7 enhancer trap (Figure S2). Finally, we modulated Wnt activity using conditional overexpression of Dkk1 or Wnt8 in heat-shocked Tg(hsp70l:dkk1b-GFP) w32 and Tg(hsp70:wnt8-GFP) w34 embryos, respectively [START_REF] Stoick-Cooper | Distinct Wnt signaling pathways have opposing roles in appendage regeneration[END_REF]. Heat-shocked embryos carrying the Tg(hsp70l:dkk1b-GFP) w32 transgene show normal expression of opn4xa (Fig 2B) suggesting that Wnt activity is dispensable for the specification of opn4xa+ cells. Similarly, heat-shocked embryos transgenic for the Tg(hsp70:wnt8-GFP) w34 transgene show normal numbers of opn4xa + cells suggesting that Wnt activity is not sufficient to drive the opn4xa+ fate. We conclude that Wnt signalling does not play a role during specification of the opn4xa+ fate.

Specification of opn4xa+ cells does not require BMP signalling and is inhibited by Notch signalling

Classical PhRs are specified by a combination of Notch and BMP pathway activity with BMP activity being necessary and sufficient to activate the PhR fate and Notch being required to inhibit the inappropriate PN program [START_REF] Cau | Notch resolves mixed neural identities in the zebrafish epiphysis[END_REF][START_REF] Quillien | BMP signaling orchestrates photoreceptor specification in the zebrafish pineal gland in collaboration with Notch[END_REF]. To understand whether opn4xa+ cells are closer to PNs or PhRs developmentally speaking, we analysed the effects of reducing BMP or Notch activities on the specification of these cells.

We used Tg(hsp70l:dnXla.Bmpr1a-GFP) w30 ; Tg(hsp70l:nog3) fr14 double transgenic embryos heat-shocked at 14hpf to reduce BMP signalling, a condition that we have previously shown reduces the number of classical PhRs [START_REF] Quillien | BMP signaling orchestrates photoreceptor specification in the zebrafish pineal gland in collaboration with Notch[END_REF].

Under these conditions, the number of opn4xa+ cells was similar compared to wildtype embryos (Fig 2A ,C); pineal opn4xa+ cells in BMP loss-of-function embryos exhibit an aberrant medial location which is likely due to the absence of classical photoreceptors normally found in the centre of the pineal gland (Fig2A). The effects of impairing Notch activity was analysed in the mindbomb (mib) mutant (mib ta52b [START_REF] Itoh | Mind bomb is a ubiquitin ligase that is essential for efficient activation of Notch signaling by Delta[END_REF]). We found that mib embryos exhibit twice the number of opn4xa+ cells at 48 hpf compared to controls (Fig 2A,D). Altogether these results suggest that pineal opn4xa+ cells behave as classical pineal PNs with respect to their requirement for BMP and Notch activity.

In contrast to classical PNs, opn4xa + projection neurons respond to light in a LIGHT ON fashion

Electrophysiological experiments performed in the rainbow trout, pike, frog and turtle have shown that projection neurons are continuously activated in the dark suggesting that they function in a LIGHT OFF mode [START_REF] Dodt | PHOTOSENSITIVITY OF THE PINEAL ORGAN IN THE TELEOST, SALMO IRIDEUS (GIBBONS)[END_REF][START_REF] Falcón | The Photosensory Function of the Pineal Organ of the Pike (Esox lucius L.) Correlation Between Structure and Function[END_REF][START_REF] Meissl | Extraocular Photoreception of the Pineal Gland of the Aquatic Turtle Pseudemys scripta elegans[END_REF][START_REF] Morita | Lead pattern of the pineal neuron of the rainbow trout (Salmo irideus) by illumination of the diencephalon[END_REF][START_REF] Morita | Nervous activity of the frog's epiphysis cerebri in relation to illumination[END_REF]. To assess whether opn4xa+ PNs are also LIGHT OFF, we analysed the expression of c-fos after a 30 minute dark-pulse in 48 hpf light-adapted embryos (Fig 3A ); c-fos is an immediate early gene that is extensively used as a read out of neuronal activation (reviewed in [START_REF] Guzowski | Mapping behaviorally relevant neural circuits with immediateearly gene expression[END_REF] . In mammals, the pineal gland is connected to the retina via a multi-synaptic pathway [START_REF] Sapède | The pineal gland from development to function[END_REF].To rule out that the pineal LIGHT ON response we describe is caused by photosensitivity in the retina, we assessed the presence of this response in lakritz (lak) mutant which lack neurotransmission from the retina owing to a complete absence of RGCs [START_REF] Kay | Retinal Ganglion Cell Genesis Requires lakritz, a Zebrafish atonal Homolog[END_REF]. The pineal LIGHT ON response is similar in lak+/+ and lak-/-embryos at 48 hpf suggesting that the retina is not involved in this activity (Fig 4D ,E).

Taken together, our results indicate heterogeneity in the light-response within the PN population with opn4xa+ PNs functioning in a LIGHT ON fashion and classical PNs in a LIGHT OFF mode.

LIGHT ON and LIGHT OFF responses are elicited by different range of wavelengths

Apart from Opn4xa, which is sensitive to blue-green light [START_REF] Davies | Functional diversity of melanopsins and their global expression in the teleost retina[END_REF], the classical PhR in the pineal express opsin photo-pigments (exorhodopsin, red cone opsin and parietopsin) that are sensitive to green and red light [START_REF] Cau | A Notch-mediated, temporal asymmetry in BMP pathway activation promotes photoreceptor subtype diversification[END_REF]. We next examined whether LIGHT ON in blue, red or green wavelengths were involved in the induction of c-fos expression. We observed that 30 minute light pulses in the blue and green range induce c-fos expression while similar pulses of red light could not induce a LIGHT ON response (Fig 5 A-C, G). Given that the absorbance spectrum of the Opn4xa photo-pigment overlaps with the emission of the green and blue LEDs used in this study, we propose that this LIGHT ON response reflects the direct photosensitivity of opn4xa+ photo-pigment. In marked contrast with the results obtained for the LIGHT ON response, LIGHT OFF was elicited when the embryos were adapted in either red, blue or green light (Figure 5 D-F, H) suggesting that this response is elicited by a wide-range of wavelengths and can, therefore, be referred to as achromatic.

Our results suggest that classical, opn4xa-projection neurons function in a LIGHT OFF mode and most likely transduce the light input received by classical PhRs.

In contrast, opn4xa+ projection neurons are activated by light and most likely directly photosensitive.

Discussion

In this paper, we describe a new population of cells in the zebrafish pineal gland. These cells express the green-blue photosensitive pigment Opn4xa and share developmental characteristics with classical, non-photosensitive projection neurons. Intriguingly, while classical PNs exhibit a LIGHT OFF response, a LIGHT ON response is observed in opn4xa+ PN upon illumination. These results suggest a previously unanticipated cellular diversity in the projection neuron population within the pineal gland. In the following section, we discuss the specification and potential function of the opn4xa+ PNs.

Projection neurons and photoreceptors of the pineal gland differ in their requirement for Notch and BMP signalling pathways: photoreceptors require BMP activation (while projection neurons do not) and BMP is in turn needed to activate the Notch pathway in these cells, which inhibits the projection neurons fate [START_REF] Cau | Notch resolves mixed neural identities in the zebrafish epiphysis[END_REF][START_REF] Quillien | BMP signaling orchestrates photoreceptor specification in the zebrafish pineal gland in collaboration with Notch[END_REF]. This led us to postulate that decoupling of the activation of BMP and Notch activity could represent an attractive mechanism to further diversify cell fates. Indeed, activation of the BMP signalling pathway without activation of Notch should lead to the generation of cells with a mixed photoreceptor/projection neuron identity [START_REF] Cau | Notch activity in the nervous system: to switch or not switch?[END_REF]. In contrast with this hypothesis, opn4xa+ cells, that exhibits both characteristics of photoreceptors and projection neurons, do not require BMP activity suggesting that they are more closely related to projection neurons than to classical photoreceptors. Similarly, Notch activity inhibits the production of both opn4xa-and opn4xa+ projection neurons to the same extent. Therefore, neither BMP nor Notch activities contribute to specifying opn4xa+ versus opn4xa-PN fates.

Since opn4xa+ and classical projection neurons show similar behaviour upon BMP or Notch loss of function, what are the signals that could specify these distinct identities? The Wnt effector tcf7 is specifically expressed in opn4xa+ PN and so is the Et(T2KHG) nns8 enhancer trap line at early stages. Nevertheless, tcf7 mutants show a normal number of opn4xa+ pineal cells. Moreover, neither gain nor loss of Wnt activity affects the number of opn4xa+ cells, which suggests that Wnt activity is neither necessary nor sufficient for specification of opn4xa expression. It remains to be addressed whether tcf7 (and/or Wnt signalling) is required to specify other aspects of the phenotype of these atypical PNs. Despite the absence of an observable function for tcf7, the Et(T2KHG) nns8 enhancer trap line provides a useful marker for opn4xa + cells at early stages which will help us in analysing the projection pattern of these cells.

Our analysis confirms previous results obtained in another teleost, the rainbow trout, as well as in numerous other species showing that an important part of the projection neuron population function in a LIGHT OFF manner [START_REF] Dodt | PHOTOSENSITIVITY OF THE PINEAL ORGAN IN THE TELEOST, SALMO IRIDEUS (GIBBONS)[END_REF][START_REF] Falcón | The Photosensory Function of the Pineal Organ of the Pike (Esox lucius L.) Correlation Between Structure and Function[END_REF][START_REF] Meissl | Extraocular Photoreception of the Pineal Gland of the Aquatic Turtle Pseudemys scripta elegans[END_REF][START_REF] Morita | Lead pattern of the pineal neuron of the rainbow trout (Salmo irideus) by illumination of the diencephalon[END_REF][START_REF] Morita | Nervous activity of the frog's epiphysis cerebri in relation to illumination[END_REF]. Within the projection neuron population only opn4xa+ PNs function in a LIGHT ON fashion. Given that this activity is only seen using blue and green light and not red light, it likely requires the function of Opn4xa, a photo-pigment with an absorbance peak at 470 nm [START_REF] Davies | Functional diversity of melanopsins and their global expression in the teleost retina[END_REF]. This result contrasts with the observation that a LIGHT ON (excitatory response) is observed at medium to long wavelengths in the rainbow trout [START_REF] Morita | Lead pattern of the pineal neuron of the rainbow trout (Salmo irideus) by illumination of the diencephalon[END_REF]. A surprising degree of variation is indeed observed in the wavelengths triggering ON and OFF chromatic responses in different species. For instance, green light triggers the excitatory response in lizards and frogs while red light elicits this response in the pike. Similarly, the chromatic inhibitory response is stronger in the ultraviolet range in fish and frogs but mainly due to blue light in lizards (see [START_REF] Dodt | The pineal and parietal organs of lower vertebrates[END_REF] for a review). It would be interesting to understand whether the LIGHT ON chromatic response observed in lizard, frog and pike require a direct photosensitivity from specific projection neurons. Alternatively, in these species, pineal photoreceptors could elicit a dual excitatory/inhibitory action on their targets similar to that described for retinal photoreceptors, which trigger a different response in bipolar cells depending on the type of glutamate receptor they express [START_REF] Euler | Glutamate responses of bipolar cells in a slice preparation of the rat retina[END_REF][START_REF] Masu | Specific deficit of the ON response in visual transmission by targeted disruption of the mGluR6 gene[END_REF][START_REF] De La Villa | L-glutamate-induced responses and cGMP-activated channels in three subtypes of retinal bipolar cells dissociated from the cat[END_REF].

Are there other LIGHT ON projection neurons in the zebrafish pineal gland? Adaptation with white light allowed us to identify an average 8 LIGHT OFF PNs for around 3 LIGHT ON PNs. Since at 48 hpf the pineal gland contains 20 PNs, either some of the PN responses have not been identified or the LIGHT OFF cells do not systematically activate c-fos following a 30 min dark pulse, in the present testing conditions. It thus remains possible that there are other unidentified projection neurons that respond in a LIGHT ON fashion for instance in response to UV light. While a complete description of the neurotransmitter/neuromodulator content of all zebrafish pineal neurons is lacking, glycinergic cells with a photoreceptor morphology have been observed in the pineal gland [START_REF] Marquart | A 3D Searchable Database of Transgenic Zebrafish Gal4 and Cre Lines for Functional Neuroanatomy Studies[END_REF][START_REF] Moly | Identification of initially appearing glycine-immunoreactive neurons in the embryonic zebrafish brain[END_REF]). These observations suggest that at least a proportion of pineal photoreceptors exert an inhibitory action on projection neurons, therefore providing a possible mechanism for the LIGHT OFF response we describe. Alternatively, photoreceptors could constantly produce an excitatory neurotransmitter in the dark, which would be blocked by light leading to an interruption of projection neuron activity through a "disfacilitation mechanism" [START_REF] Uchida | Signal transmission from pineal photoreceptors to luminosity-type ganglion cells in the lamprey, Lampetra japonica[END_REF]. A more detailed analysis of the neurotransmitter/neuromodulator content of all pineal neurons, and opn4xa+ PNs in particular, would be useful. Finally, the fact that the LIGHT OFF response is observed independently of the wavelength at which the embryos are adapted suggests that LIGHT OFF projection neurons receive inputs from several types of pineal photoreceptors.

Although direct photosensitivity of opn4xa+ PNs remains to be confirmed, they share many characteristics with retinal ipRGCS raising intriguing questions regarding the functions of these pineal cells. Indeed, in mammals ipRGCs exhibit crucial roles during the control of circadian, wake/sleep rhythms as well as in mediating the direct effects of illumination on physiology and behaviour (see Lazzerini [START_REF] Lazzerini Ospri | Mood, the Circadian System, and Melanopsin Retinal Ganglion Cells[END_REF]; [START_REF] Lucas | How rod, cone, and melanopsin photoreceptors come together to enlighten the mammalian circadian clock[END_REF] for reviews)). In particular, ipRGCs transmit light information, via their own intrinsic photosensitivity as well as inputs from rod and cone PhRs, to the mammalian 'master clock' the suprachiasmatic nucleus [START_REF] Hannibal | Melanopsin containing retinal ganglion cells are light responsive from birth[END_REF][START_REF] Hattar | Melanopsincontaining retinal ganglion cells: architecture, projections, and intrinsic photosensitivity[END_REF][START_REF] Hattar | Central projections of melanopsin-expressing retinal ganglion cells in the mouse[END_REF]. It is unclear whether a suprachiasmatic nucleus is present in the larval zebrafish and whether opn4xa+ retinal or pineal cells project onto such a structure. On the other hand, the zebrafish pineal gland has long been thought to play a 'master clock' role owing to its direct photosensitivity, its capacity to generate intrinsic rhythms of melatonin synthesis/secretion and the fact that disrupting the function of the molecular clock in pineal PhRs affect circadian rhythms of locomotor activity (Ben-Moshe [START_REF] Livne | Genetically Blocking the Zebrafish Pineal Clock Affects Circadian Behavior[END_REF][START_REF] Bolliet | Photoreceptor cells of the pike pineal organ as cellular circadian oscillators[END_REF][START_REF] Cahill | Circadian regulation of melatonin production in cultured zebrafish pineal and retina[END_REF][START_REF] Cahill | Circadian melatonin rhythms in cultured zebrafish pineals are not affected by catecholamine receptor agonists[END_REF][START_REF] Falcón | Regulation of melatonin secretion in a photoreceptive pineal organ: an in vitro study in the pike[END_REF]. Could opn4xa+ PNs play a role alongside classical pineal PhRs in the function of the circadian system? Analysis of an opn4xa mutant allele combined with retina-specific and/or pineal specific transgenic tools would help us understand whether opn4xa+ PNs are important players in this field.

Experimental Procedures

Zebrafish lines and developmental conditions All animals were handled in the CBI fish facility, which is certified by the French Ministry of Agriculture (approval number A3155510). The project was approved by the French Ministry of Teaching and Research (agreement number APAFIS#3653-2016011512005922).

Embryos were reared at 28 degrees in a 14 hours Light/ 10 hours Dark cycle and staged according to standard protocols. All the transgenic and mutant lines have been described previously: Tg(aanat2:GFP) y8 [START_REF] Gothilf | Pineal-specific expression of green fluorescent protein under the control of the serotonin-N-acetyltransferase gene regulatory regions in transgenic zebrafish[END_REF], Tg(elavl3:EGFP) knu3 [START_REF] Park | Analysis of upstream elements in the HuC promoter leads to the establishment of transgenic zebrafish with fluorescent neurons[END_REF], Et(T2KHG) nns8 [START_REF] Nagayoshi | Insertional mutagenesis by the Tol2 transposon-mediated enhancer trap approach generated mutations in two developmental genes: tcf7 and synembryn-like[END_REF], Tg(hsp70l:dkk1b-GFP) w32 and Tg(hsp70:wnt8-GFP) w34 [START_REF] Stoick-Cooper | Distinct Wnt signaling pathways have opposing roles in appendage regeneration[END_REF], Tg(hsp70l:dnXla.Rbpj-MYC) vu21 and Tg(hsp70l:nog3) fr14 [START_REF] Chocron | Zebrafish Bmp4 regulates left-right asymmetry at two distinct developmental time points[END_REF] , mib ta52b [START_REF] Itoh | Mind bomb is a ubiquitin ligase that is essential for efficient activation of Notch signaling by Delta[END_REF], lakritz [START_REF] Kay | Retinal Ganglion Cell Genesis Requires lakritz, a Zebrafish atonal Homolog[END_REF]. Conditions of heat-shock were: Tg(hsp70l:dnXla.Rbpj-MYC) vu21 and Tg(hsp70l:nog3) fr14 : 30 minutes at 39.5°C; Tg(hsp70l:dkk1b-GFP) w32 and Tg(hsp70:wnt8-GFP) w34 : 1 hour at 38°C. The protocol for genotyping lakritz embryos is available upon request.

Application of different light regimes

To assess LIGHT ON responses, two different protocols were used. In the first protocol, the light was turned on at 9 am and the embryos fixed at 9.30 am. In the second one, the embryos were dark adapted for 2 hours (from 9.30 to 11.30) before a 30 mn light pulse and a fixation at 12 am. We did not notice a difference in the responses between these two protocols. On the other hand, for the assessment of the LIGHT OFF responses, the embryos were light-adapted from 9 am to 12 am after which light was turned off and the embryos were fixed 30 mns after. The comparison of adapted and pulsed embryos fixed at the same circadian time allowed us to conclude that the observed c-fos expression was caused by the pulse rather than by circadian rhythms.

Illumination with white light was performed using a regular neon lamp (80 lux).

Illumination with blue, green and red light was performed in a MWP unit (Zantiks) equipped with standard blue, green and red LEDs. The characteristics of the light emitted from these LEDS is: Blue light: 92 lux, max= 449nm, half-band width: 25,4nm

Green light: 109 lux, max= 512 nm, half-band width: 33,07 nm Red light: 34 lux, max= 627 nm, half-band width: 21,67 nm Scripts controlling the different light regimes are available upon request.

Immunohistochemistry and in situ hybridisation

Immunohistochemistry and in situ hybridisation were performed as previously described and fluorescent in situ hybridisation were revealed using either Fast Red (Sigma) or the TSA Plus System (TSA-Fluorescein, Perkin Elmer; [START_REF] Cau | A Notch-mediated, temporal asymmetry in BMP pathway activation promotes photoreceptor subtype diversification[END_REF].

Immunohistochemistry against Tcf7l2 was performed as described in [START_REF] Hüsken | Tcf7l2 is required for left-right asymmetric differentiation of habenular neurons[END_REF] using an anti-Human TCF3,4 antibody (1:400, Biomol, clone 0.T.148).

Probes used in this study were: opn4xa [START_REF] Matos-Cruz | Unexpected diversity and photoperiod dependence of the zebrafish melanopsin system[END_REF], c-fos [START_REF] Ellis | Distinct models of induced hyperactivity in zebrafish larvae[END_REF], lef1 and tcf7 [START_REF] Veien | Expression pattern of zebrafish tcf7 suggests unexplored domains of Wnt/beta-catenin activity[END_REF]. (C-K) Co-expression of opn4xa mRNA (in red) on the one hand with the Tg(aanat2:GFP) y8 ('aanat2:GFP', C,D,E), the Tg(elavl3:EGFP) knu3 ('elavl3:EGFP', F,G,H) and the Et(T2KHG) nns8 ('tcf7:GFP', I,J,K transgenes (in green). (B) Effect of gain or loss of Wnt activity on the number of pineal opn4xa+ cells: Embryos from Et(T2KHG) nns8 +/-incrosses were assessed for GFP fluorescence; countings were performed at 30hpf. The size of the GFP-and GFP+ populations ('tcf7:GFP-' and 'tcf7:GFP+') were respectively n=8 and n=31. Embryos carrying the Tg(hsp70l:dkk1b-GFP) w32 transgene after an heat shock at 18 hpf, were compared with negative siblings at 48 hpf (n=13 for the 'hs:dkk1-' and n=11 for the 'hs:dkk1+'). Embryos carrying the Tg(hsp70:wnt8-GFP) w34 transgene (n=14) after an heat shock at 14 hpf, were compared to siblings (n=14) at 48 hpf.

Cell counting and statistical Analysis

  . While light-adapted embryos fixed at the same circadian time show no c-fos expression in the pineal (Fig 3Bi), a pulse of dark induced expression of c-fos expression in 8.36±2.13 cells per embryo, which are located laterally in the pineal (from n=14 embryos) (Fig 3Bii). The same experiment performed in Et(T2KHG) nns8 or Tg(elavl3:EGFP) knu3 embryos showed that these LIGHT OFF cells are tcf7:GFP-but elavl3:EGFP+ and thus correspond to classical, opn4xa-projection neurons (Fig 3C,D). To address if opn4xa+ PNs are LIGHT ON, next we examined the effect of a light pulse in dark-adapted embryos (Fig 4A). We found that a 30 minute pulse of white light delivered to 48 hpf dark-adapted embryos induced c-fos expression in 3±1.41 cells (from n=17 embryos) located laterally within the pineal organ (Fig 4Bii) in cells that strongly express the Et(T2KHG) nns8 transgene (Fig 4C); c-fos transcript was not detected in the pineal of non-light stimulated embryos fixed at the same circadian time (Fig 4Bi). Similar results were obtained in 27 hpf and 3 dpf dark-adapted embryos (Fig S3)

Fluorescent

  cells were counted using the ImageJ software on confocal data sets with 2 m interval optical planes. Statistical tests and number of embryos used are stated in each figure and/or figure legend.

Figure 1 :

 1 Figure 1: opn4xa is expressed in a restricted population of projection neurons

  (A-K) Anterior is upwards. Scale bars: 10 µm. (L)Time course of opn4xa expression in the developing pineal organ. Average numbers of opn4xa+ cells are quantified at different time points indicated in hours postfertilisation (hpf). Values are mean ±SD, n=4, 5, 3, 6, 9, 26, 6 and 6, respectively.

Figure 2 :

 2 Figure 2: Effect of manipulating Notch, BMP and Wnt pathways on the

  (C) Effect of a reduction in Notch activity: Control embryos (n=30) were compared to homozygous mindbomb (mib-/-; n=7) mutants at 48 hpf (D) Effect of a reduced BMP signalling: Embryos transgenic for Tg(hsp70l:dnXla.Bmpr1a-GFP) w30 (hs:dnBMPR1a, n=12) or Tg(hsp70l:nog3) fr14

(

  hs:noggin3, n=30) or both (n=18) were compared to siblings (n=7). Kruskal-Wallis test with Dunn's multiple comparisons test, P=0.44; not significant.

Figure 3 :

 3 Figure 3: Identification of a population of opn4xa-PNs functioning in a LIGHT

Figure 4 :

 4 Figure 4: opn4xa+ PNs function in a LIGHT ON mode:

Figure 5 :

 5 Figure 5: Different spectral sensitivities for the LIGHT ON and the LIGHT OFF

Acknowledgements

We thank S. Mazères for his help with the spectral analysis of LED light, R. Dorsky for providing the Et(T2KHG) nns8 , Tg(hsp70l:dkk1b-GFP) w32 and Tg(hsp70:wnt8-GFP) w34 transgenic lines, and M. Halpern and K. Soanes for sharing probes. We are indebted to members of the Blader team for helpful discussions. This work was supported by the Centre National de la Recherche Scientifique (CNRS); the Institut National de la Santé et de la Recherche Médicale (INSERM); Université de Toulouse III (UPS); Fondation pour la Recherche Médicale (FRM; DEQ20131029166); Fédération pour la Recherche sur le Cerveau (FRC); Association pour la Recherche sur le Cancer (ARC); Association Rétina France and the Ministère de la Recherche. We would like to thank Brice Ronsin, Stéphanie Bosch and the Toulouse RIO Imaging platform; as well as Stéphane Relexans, Aurore Laire and Richard Brimicombe for taking care of the fish.

Declaration of competing interest

The authors declare that no competing interests exist.

Figure and


Sarah Komaty

Hayriye Özçelik

Moussa Zaarour

Aurélie Ferre

Samuel Valable
email: samuel.valable@cnrs.fr

Svetlana Mintova
email: svetlana.mintova@ensicaen.fr

Ruthenium tris bipyridyl complex encapsulated in nanosized FAU zeolite as intracellular localization tracer

Keywords: Zeolites, nanocrystals, encapsulation, luminescence, intracellular tracer, cell uptake, cytotoxicity

Designing zeolites for medical applications is a challenging task requiring the introduction of new functionalities without altering their intrinsic properties such as morphology, crystallinity, colloidal stability, surface charge, and porosity. Herein, we present the encapsulation of luminescent ruthenium-tris(2,2′-bipyridyl) complex in FAU zeolite nanocrystals (Ru(bpy)3-FAU) and their use as an intracellular localization tracer. Upon exciting of the Ru(bpy)3-FAU zeolite at 450 nm, the sample gives rise to an orange-red emission at 628 nm thus permitting its use for cellular imaging and localization of the zeolite nanoparticles. The nanosized Ru(bpy)3-FAU zeolite is characterized in terms of size, charge, crystallinity, morphology, porosity, thermal stability, and sorption capacity. The potential toxicity of Ru(bpy)3-FAU on U251-MG glioblastoma cells was evaluated. A safe concentration (50-100 µg/ml) for the Ru(bpy)3-FAU zeolite is identified. The luminescent properties of the ruthenium complex confined in the zeolite nanocrystals allow to detect their localization in the U251-MG cells with a main accumulation in the cytoplasm. The Ru(bpy)3-FAU nanosized zeolite is a potential candidate for biological applications for being stable, safe, capable of loading respiratory gases, and easily probed in the cells owing to its luminescent properties.

INTRODUCTION

Zeolites are crystalline microporous aluminosilicates with large surface area, and well-defined pores and cages. The size and shape of their pores and cages are the base for the selective gas sieving and discrimination of guest molecules. They allow ions and compounds with smaller dimensions than the zeolite pore apertures to diffuse into the internal voids, whereas they restrict the access for bigger compounds and complexes. The zeolite internal voids are usually occupied with charge compensating cations and water molecules; these can be exchanged for other cations or partially replaced with appropriate adsorbing compounds. [START_REF] Zaarour | Progress in Zeolite Synthesis Promotes Advanced Applications[END_REF] The admissibility of zeolites for various applications is linked to their modifiable chemical composition, high porous character, exchangeable charge compensating cations, variable hydrophilicity/hydrophobicity, and controlled adsorption properties. Recently, more attention was given to the use of zeolites in biomedical applications [START_REF] Bacakova | Applications of Zeolites in Biotechnology and Medicine -a Review[END_REF] as antimicrobial agents, [START_REF] Anfray | Acute Toxicity of Silver Free and Encapsulated in Nanosized Zeolite for Eukaryotic Cells[END_REF][START_REF] Dong | Silver Confined within Zeolite EMT Nanoparticles: Preparation and Antibacterial Properties[END_REF] anti-cancer drugs carriers, [START_REF] Vilaça | Potentiation of 5-Fluorouracil Encapsulated in Zeolites as Drug Delivery Systems for in Vitro Models of Colorectal Carcinoma[END_REF] and in the storage and controlled delivery of drugs and gases. [START_REF] Seifu | Tissue Engineering Scaffolds Containing Embedded Fluorinated-Zeolite Oxygen Vectors[END_REF][START_REF] Komaty | A Facile Route toward the Increase of Oxygen Content in Nanosized Zeolite by Insertion of Cerium and Fluorinated Compounds[END_REF][START_REF] Tavolaro | Zeolite Inorganic Supports for BSA Immobilization: Comparative Study of Several Zeolite Crystals and Composite Membranes[END_REF] More specifically, they were considered as carriers for the delivery of anti-neoplastic compounds, [START_REF] Datt | Loading and Release of 5-Fluorouracil from HY Zeolites with Varying SiO2/Al2O3 Ratios[END_REF][START_REF] Spanakis | Controlled Release of 5-Fluorouracil from Microporous Zeolites[END_REF][START_REF] Grund | Mitoxantrone-Loaded Zeolite Beta Nanoparticles: Preparation, Physico-Chemical Characterization and Biological Evaluation[END_REF] nucleic acids, [START_REF] Lülf | Multifunctional Inorganic Nanocontainers for DNA and Drug Delivery into Living Cells[END_REF][START_REF] Bertucci | Intracellular Delivery of Peptide Nucleic Acid and Organic Molecules Using Zeolite-L Nanocrystals[END_REF] both hydrophilic [START_REF] Fatouros | In Vitro and in Silico Investigations of Drug Delivery via Zeolite BEA[END_REF][START_REF] Paradee | Encapsulation of Folic Acid in Zeolite y for Controlled Release via Electric Field[END_REF] and lipophilic drugs, [START_REF] Datt | Loading and Release of 5-Fluorouracil from HY Zeolites with Varying SiO2/Al2O3 Ratios[END_REF][START_REF] Karavasili | Dissolution Enhancement of the Poorly Soluble Drug Nifedipine by Co-Spray Drying with Microporous Zeolite Beta[END_REF] anti-inflammatory drugs, [START_REF] Karavasili | Comparison of Different Zeolite Framework Types as Carriers for the Oral Delivery of the Poorly Soluble Drug Indomethacin[END_REF] and also for the delivery of gases that could be of interest in the biomedical field. [START_REF] Komaty | A Facile Route toward the Increase of Oxygen Content in Nanosized Zeolite by Insertion of Cerium and Fluorinated Compounds[END_REF][START_REF] Komaty | Incorporation of Trivalent Cations in NaX Zeolite Nanocrystals for the Adsorption of O2 in the Presence of CO2[END_REF] Faujasite nanozeolite is a potential candidate for biomedical applications due to its large surface area, high micropore volume, and the presence of supercage (1.2 nm) that can host different drugs [START_REF] Braschi | Removal of Sulfonamide Antibiotics from Water: Evidence of Adsorption into an Organophilic Zeolite Y by Its Structural Modifications[END_REF][START_REF] Zhang | Controlled Release of Paraquat from Surface-Modified Zeolite Y[END_REF] or gases. [START_REF] Tavolaro | Zeolite Inorganic Supports for BSA Immobilization: Comparative Study of Several Zeolite Crystals and Composite Membranes[END_REF] Besides, zeolites modified with luminescent metal clusters, [START_REF] Dong | Sodalite Cages of EMT Zeolite Confined Neutral Molecular-like Silver Clusters[END_REF] lanthanides, [START_REF] Wang | Luminescent Materials of Zeolite Functionalized with Lanthanides[END_REF] and metal complexes [START_REF] Ruda-Eberenz | Entrapment of Ionic Tris(2,2′-Bipyridyl) Ruthenium(II) in Hydrophobic Siliceous Zeolite: O 2 Sensing in Biological Environments[END_REF] were also used to upgrade their properties especially for optical oxygen sensors application. Confining luminescent compounds within the zeolites limits the formation of chromophore aggregates. Consequently, partial or complete loss of emission through nonradiative pathways [START_REF] Busby | Interactions of Perylene Bisimide in the One-Dimensional Channels of Zeolite L[END_REF] is eliminated, and the luminescent properties are enhanced. [START_REF] Calzaferri | Host-Guest Antenna Materials[END_REF][START_REF] Alarcos | Photochemistry and Photophysics in Silica-Based Materials: Ultrafast and Single Molecule Spectroscopy Observation[END_REF] Numerous nanoparticles enter inside the cells through a process termed "endocytosis". The particles immersed in membrane-bound vesicles are also known as endosomes (or phagosomes in case of phagocytosis), [START_REF] Iversen | Endocytosis and Intracellular Transport of Nanoparticles: Present Knowledge and Need for Future Studies[END_REF][START_REF] Sahay | Endocytosis of Nanomedicines[END_REF] which are pinched off membrane invaginations. Then the cargo is sorted through vesicles to various intracellular compartments or nuclei and recycled to extracellular environment. [START_REF] Iversen | Endocytosis and Intracellular Transport of Nanoparticles: Present Knowledge and Need for Future Studies[END_REF][START_REF] Sahay | Endocytosis of Nanomedicines[END_REF] There, the guest molecule would allow monitoring the exact location of the nanoparticles (zeolites or others) owing to their luminescent properties. Most studies of the cellular uptake and tracing of nanoparticles have been performed using luminescent labelled nanoparticles. [START_REF] Snipstad | Labeling Nanoparticles: Dye Leakage and Altered Cellular Uptake[END_REF] Experiments revealed several problems associated with such systems, especially in the case of organic dyes; photodecomposition, insolubility in the hydrophobic support matrices, and leaching from the zeolite support are often observed. A solution to these problems is to confine a luminescent molecule such as ruthenium-tris(2,2′bipyridyl) within zeolite nanoparticles, for example in the supercages of Faujasite (FAU) zeolite.

The diameter of the ruthenium-tris(2,2′-bipyridyl) complex is 12 Å, which is comparable to the size of the supercage of FAU. However, the diameter of the ruthenium-tris(2,2′-bipyridyl) complex is by far larger than the pore opening of zeolite (7.4 Å), thus cannot leach out of the pores. [START_REF] Payra | Development of a Dissolved Oxygen Sensor Using Tris(Bipyridyl) Ruthenium (II) Complexes Entrapped in Highly Siliceous Zeolites[END_REF] The use of modified FAU as optical sensor was reported in literature. [START_REF] Meier | Novel Oxygen Sensor Material Based on a Ruthenium Bipyridyl Complex Encapsulated in Zeolite Y: Dramatic Differences in the Efficiency of Luminescence Quenching by Oxygen on Going from Surface-Adsorbed to Zeolite-Encapsulated Fluorophores[END_REF] Additionally, the migration of oxygen within zeolite by studying its emission quenching, [START_REF] Coutant | Oxygen Transport in Zeolite Y Measured by Quenching of Encapsulated Tris(Bipyridyl)Ruthenium[END_REF] and detection of dissolved oxygen in aqueous media were also reported. [START_REF] Dong | Sodalite Cages of EMT Zeolite Confined Neutral Molecular-like Silver Clusters[END_REF][START_REF] Ruda-Eberenz | Entrapment of Ionic Tris(2,2′-Bipyridyl) Ruthenium(II) in Hydrophobic Siliceous Zeolite: O 2 Sensing in Biological Environments[END_REF] Herein, we present the incorporation of ruthenium-tris(2,2′-bipyridyl) complex in nanosized FAU zeolite crystals with a particle size in the range of 20-30 nm (sample Ru(bpy)3-FAU). The nanosized FAU zeolite containing the ruthenium complex, due to the optical properties inherited from the luminescent ruthenium complex is used as an intracellular localization tracer. The cytotoxicity tests of Ru(bpy)3-FAU for eukaryotic cells reveal that it is safe in the concentration range of 50-100 µg/ml. Due to its small dimensions, the modified zeolite crystals are capable of crossing the cell membrane barrier and residing in the cytoplasm.

Owing to all these features, we believe that the Ru(bpy)3-FAU nanocrystals can be used as a safe intracellular localization tracer and active carrier for various biomedical uses.

Experimental part

Materials: Al powder (Al, 325 mesh, 99.5%, Alfa Aesar, Karlsruhe, Germany), sodium hydroxide (NaOH, 97%, Sigma-Aldrich, Lyon, France), colloidal silica (SiO2, Ludox HS-30, 30 wt % SiO2, pH = 9.8, Sigma-Aldrich, Lyon, France), ruthenium chloride (99.9% (PGM basis), Ru 38% min, Alfa Aesar, Karlsruhe, Germany), 2,2'-dipyridyl (99+ %, Acros Organics), N,Ndimethylformamide (DMF, 99.8+%, Alfa Aesar, Karlsruhe, Germany), lithium chloride, (99+%, Thermo Fisher Scientific, Geel, Belgium), acetonitrile anhydrous (ACROS Organics), ethanol absolute (Sigma-Aldrich, Lyon, France), and diethyl ether (99 +%, ACROS Organics) were used as received.

A human glioblastoma cell line, U251-MG was purchased from American Type Culture Collections (ATCC, Manassas, VA, USA). Dulbecco's Modified Eagle Medium (DMEM), (Low Glucose), penicillin/streptomycin (10,000 units), trypsin-EDTA (0.25%), glutamine, Paraformaldehyde, Hoechst 33342 (10 µg/ml) were purchased from Sigma, France.

Preparation of zeolite X (sample FAU):

The nanosized FAU zeolite in sodium form free of organic template (20-30 nm) with Si / Al = 1.2 was-prepared following an original reported procedure by our group. [START_REF] Awala | Template-Free Nanosized Faujasite-Type Zeolites[END_REF] Encapsulation of ruthenium-tris(2,2′-bipyridyl) in nanosized FAU zeolite (sample Ru(bpy)3-FAU): Ru(bpy)3-FAU was-prepared by following a reported procedure after modification. [START_REF] Busby | Interactions of Perylene Bisimide in the One-Dimensional Channels of Zeolite L[END_REF] One molar equivalence (1 eq) of RuCl3 (0.128g), 2 eq of LiCl (0.06 g), and 1 eq of 2.2'-bipyridyl (0.096 g) were introduced into 5 mL of DMF. The solution was heated under stirring at 90 ºC for 7 h and then left to cool down to room temperature. The obtained product was purified by crystallization in diethyl ether, dissolved in CH3CN, and then passed over Büchner funnel to eliminate LiCl. The obtained red solution was added under N2 atmosphere to 300 mg of FAU readily preheated at 200 ºC under vacuum for 2 h; the resulting suspension was stirred for 18 h at room temperature. At the end of the reaction, the powder was collected by centrifugation (20000 rpm, 30 min) and washed several times with acetonitrile until the supernatant became colorless. The collected brownish zeolite was dispersed in 50 mL of ethanol, and then an excess of the ligand 2,2'-bipyridyl (0.528 g) was introduced. The mixture was refluxed for two days during which the color evolved from green to brownish -red to orange. The product was separated by centrifugation, washed three times by acetonitrile and two times by ethanol using centrifugation (20000 rpm, 30 min).

The product, the nanosized FAU zeolite containing ruthenium complex (Ru(bpy)3-FAU), is used as solid and colloidal suspension (distilled water as solvent) for further characterization.

Characterization Dynamic light scattering (DLS) and zeta potential:

The size of the nanoparticles in water suspension was measured by a Malvern Zetasizer Nano instrument using a backscattering geometry (scattering angle of 173°, He-Ne laser with a 3 mW output power at a wavelength of 632.8 nm). The DLS analyses were performed on water suspensions at a solid concentration of 1 wt %. The surface charge of the crystals was determined by measuring the zeta potential value of water suspensions at a constant solid concentration of 1 wt %.

X-ray diffraction (XRD):

The crystallinity of the powder samples (FAU and Ru(bpy)3-FAU) was evaluated using a PANalyticalX'Pert Pro diffractometer with a CuK monochromatized radiation ( = 1.5418 Å).

N2 adsorption:

The porosity of the samples was measured using Micrometrics ASAP 2020 volumetric adsorption analyzer. Samples were degassed at 275 C under vacuum overnight before the measurement. The external surface area and micropore volume were estimated by the alpha-plot method using Silica-1000 (22.1 m 2 g -1 assumed) as a reference. The micropore and mesopore size distributions of solids were estimated by the Nonlocal Density Functional Theory (NLDFT) and Barret-Joyner-Halenda (BJH) methods using the desorption branch, respectively.

Thermogravimetric analysis (TGA):

The amount of ruthenium-tris(2,2′-bipyridyl) and moisture in the samples were investigated using SETSYS instrument (SETARAM) analyzer.

Samples were heated with 5 °C min -1 rate under 40 ml.min -1 flow of air.

In situ Fourier Transform Infrared Spectroscopy (FTIR): Zeolite powders were pressed (~10 7 Pa) into self-supported disks (2 cm 2 area, 20 mg•cm -2 ) and placed in an IR cell equipped with KBr windows. IR spectra were recorded using a Nicolet 6700 IR spectrometer (Thermo Scientific, Villebon sur Yvette, France) equipped with a mercury cadmium telluride (MCT)

detector and an extended KBr beam splitter. Spectra were recorded in the 400-5500 cm -1 range at 4 cm -1 with 128 scans. A homemade IR cell was used to evacuate the samples; then samples were heated up to 200 °C for 2 h under vacuum before the measurements. Various amounts of oxygen (0-100 Torr) and carbon dioxide (0-20 Torr) were introduced into the cell and kept in equilibrium for 5 min at -196 °C in the case of O2, and for 1 min at room temperature in the case of CO2 before recording each spectrum. The zeolites were loaded with increasing amounts of O2 and CO2 until their corresponding FTIR bands reached saturation. To allow the comparison of different samples, the spectra were normalized to the samples' mass and plotted as absorbance per gram over the wavelength

UV-vis absorption spectroscopy:

The UV-vis absorption spectra of the zeolite suspensions were recorded on a Varian Cary 4000 UV-vis spectrometer in transmission mode using a quartz cuvette with a 1 cm path length. The as-synthesized zeolite (FAU) without Ru(bpy)3 was used as a reference.

Excitation and luminescence study: The excitation and emission spectra of the diluted Ru(bpy)3-X zeolite sample (50 µg/mL diluted 10x with dH2O in 96-well plates) were recorded using Spark microplate reader (Tecan Group Ltd., Switzerland).

Cell culture: U251-MG (Sigma-Aldrich) human glioblastoma cells were cultured in Dulbecco's Modified Eagle Medium DMEM (1x) with 10% of Fetal Bovine Serum, 1% of Penicilin-Streptomycin, and 1% glutamin. The cell line was maintained under a humidified atmosphere containing 5% CO2 at 37 º C.

Cell viability assay: Crystal violet assay followed by spectrophotometric assay were performed to assess the effect of Ru(bpy)3-FAU on cell viability. For this purpose, U251-MG cells were seeded into 96-well plates at the concentration of 7500 cells/well and incubated overnight at 37 º C under 5% CO2 atmosphere. Then the media was replaced by zeolite suspensions with increasing the concentrations of 50-100-250-500 µg/mL, and incubated for 1 day, 4 days and 7 days. At each time point, the culture medium was removed from wells and the plate was washed with warmed Phosphate Buffered Saline (PBS). Crystal violet dye solution (500 µL) was added into each well and the plate was incubated for 30 minutes at room temperature; the plate was washed with water and dried. The crystal violet dye was solubilized by adding 1 mL of 10 % acetic acid.

The plate was agitated on orbital shaker until the color became uniform with no areas of dense coloration in the bottom of wells. The absorbance at 600 nm in a microplate reader was measured. In order to verify the density of the cells after staining, light microscopy images were taken by Nikon inverted microscope.

Confocal microscopy analysis:

The cell uptake of Ru(bpy)3-FAU into U251-MG cells was investigated by confocal microscopy. The cells were seeded onto coverslips in 24-well plates with a density of 2x10 4 cells per well, and incubated at 37ºC in 5% CO2 atmosphere overnight.

Afterwards, 50 µg/mL of the zeolite was added and incubated for 3 days. At the end of the incubation period, the cells were rinsed with PBS and fixed with 4% paraformaldehyde for 15 min. After washing with PBS, nuclei were stained with Hoechst 33342 (10 µg/ml) for 10 min. In the next step, the samples washed two times with PBS (5 min each) and the cells were mounted.

Confocal images with a 60x immersion oil objective were obtained with an Olympus FV1000 confocal microscope (Olympus). Excitation and emission spectra of markers Hoescht 361/497 nm, Ru(bpy)3-FAU 488/543 nm. The images were edited using FV1000 software (Olympus) and Fiji/Image J. 34

Results and Discussion

Physicochemical characterization of nanosized Ru(bpy)3-FAU zeolite

The structure and crystallinity of the as-synthesized and the modified FAU zeolite after encapsulating ruthenium-tris(2,2′-bipyridyl) are studied by X-ray diffraction. The XRD pattern of the Ru(bpy)3-FAU zeolite perfectly matches that of the as-synthesized FAU zeolite (Figure 1).

Moreover, the broad Bragg peaks recorded for both samples suggest the presence of zeolite crystals with small dimensions. The size of the crystals in the samples was further characterized by DLS (Figure 2A). Very small discrete crystals with an average size of 20-30 nm were unveiled; a monomodal particle size distribution curve was measured with no evidence of aggregation. Confining the ruthenium complex within the zeolite nanocrystals did not impact its stability in the colloidal suspensions using water as a solvent. The as-synthesized FAU and Ru(bpy)3-FAU zeolite samples exhibited high negative zeta potential values of -51 mV and -42 mV, respectively that confirms the negative surface charge of the crystals and high stability (Figure 2B). Thermogravimetric analyses (TGA) revealed that the as-prepared FAU zeolite loses 23% of its mass at 79 °C and 120 °C. These losses are attributed to the release of H2O molecules from the surface and from the channels of the zeolite crystals (Figure 3). A different TG profile was recorded for the Ru(bpy)3-FAU sample; a single mass loss at 120 °C was measured that correspond to H2O with lower quantity (17%) than the parent FAU sample. This decrease of water content can be explained by the partial replacement of water with the ruthenium complex.

The ruthenium complex was released from the Ru(bpy)3-FAU sample in the temperature range of 280-500 °C contributing to 8 % of the total mass loss (Figure 3). The ruthenium-tris(2,2′bipyridyl) complex was eliminated from the zeolite in a step-wise manner exemplified by the appearance of 4 mass losses at high temperatures: two overlapping bands in the range of 280-350 °C, one band at 395 °C, and two partially overlapping bands at high temperature (470-500 °C)

(Figure 3Aa and3Ba). The presence of ruthenium-tris(2,2′-bipyridyl) is further confirmed by FTIR. The characteristic bands at 1600 (ν25), 1486 (ν18+ν33), 1464 (ν27), 1448 (ν18+γCH), and 1427 cm -1 (ν28) resembling those of bypyridyl ligand are present (Figure S1). The successful incorporation of the ruthenium complex in the FAU is also proved by N2 sorption analyses (Figure 4). A remarkable decrease in BET specific surface area, SBET (22%) and micropore volume (28 %) in comparison to the parent FAU zeolite was measured for the Ru(bpy)3-FAU sample (Table 1). In contrast, a negligible decrease in the external surface area less than < 3 % was observed. These results indicate that the ruthenium complex is mainly Dissociation of Ru(bpy) 3 complex localized in the internal voids and can be occasionally found on the pore openings, but it is not present on the surface of FAU zeolite crystals. The incorporation of ruthenium-tris(2,2′-bipyridyl) complex in the Ru(bpy)3-FAU zeolite sample was further validated by UV-vis spectroscopy (Figure 5A). A typical UV-vis spectrum of Ru(bpy)3 2+ was recorded which contains (i) an intense band at 285 nm attributed to the π-π* ligand-center (LC) transitions, (ii) two shoulders around 325 and 345 nm corresponding to the dd metal-centered (MC) transitions, and (iii) a band at 240 nm together with a broad band extending from 380 nm to 500 nm in the visible region attributed to the metal to ligand charge transfer (MLCT) transitions from the d-orbitals of the Ru metal center to the π* orbitals of the pyridyl rings. [START_REF] Kober | Concerning the Absorption Spectra of the Ions M(Bpy)32+ (M = Fe, Ru, Os; Bpy = 2,2'-Bipyridine)[END_REF][START_REF] Juris | II) Polypyridine Complexes: Photophysics, Photochemistry, Eletrochemistry, and Chemiluminescence[END_REF][START_REF] Kalyanasundaram | Photophysics, Photochemistry and Solar Energy Conversion with Tris(Bipyridyl)Ruthenium(II) and Its Analogues[END_REF][START_REF] Kirketerp | Absorption Spectrum of Isolated Tris(2,2′-Bipyridine)Ruthenium(II) Dications in Vacuo[END_REF] Considering that the sample was thoroughly washed before the measurement, all non-reacted species or loosely adsorbed complexes are eliminated. Consequently, the recorded spectrum corresponds only to the species associated with the zeolite. The same absorption features were also recorded for the Ru(bpy)3-FAU powder (Figure S2), thus confirming the association of these absorption features to the Ru(bpy)3-FAU zeolite sample.

The excitation spectrum of the sample Ru(bpy)3-FAU shows a band centered at 450 nm (Figure 6B) resembling the MLCT band of the absorption profile (Figure 5A). This observation suggests that the low-lying MLCT is the emissive transition the Ru(bpy)3-FAU zeolite. Indeed, upon exciting at 450 nm, an orange-red emission at 628 nm is generated (Figure 5B). This emission is characterized by a structureless broad band, large stock shift from the excitation wave length (~170 nm), and large redshift from the emission wavelength of the free bipyrinde ligand (423 nm). [START_REF] Juris | II) Polypyridine Complexes: Photophysics, Photochemistry, Eletrochemistry, and Chemiluminescence[END_REF] These findings confirm the attribution of luminescence to 3 MLCT. Such feature permits the use of Ru(bpy)3-FAU zeolite as an intracellular localization tracer agent in cellular imaging and for localizing the zeolite nanocrystals.

Further the CO2 adsorption capacity of the as-prepared FAU and Ru(bpy)3-FAU samples were studied by in situ FTIR spectroscopy. The CO2 loaded in a carrier such as nanoparticles is highly desirable for controlled release into the vessels provoking blood dilatation. The FTIR spectra for both samples before and after delivery of CO2 (1 Torr for FAU and 20 Torr for Ru(bpy)3-FAU) are depicted in Figure S3. After the CO2 adsorption, the spectra exhibit a new band at 2345 cm -1 corresponding to physisorbed CO2 and a set of bands in the region 1725 cm -1 to 1350 cm -1 associated with the chemisorbed CO2 species. On the other hand, the chemisorbed CO2 or carbonates are produced following the chemical interaction between the extra framework cations in the FAU zeolite and CO2, these bonds are stable and cannot release CO2 at the body temperature. The FTIR bands of the chemisorbed species partially overlap with those of the ruthenium complex, which prevents their accurate quantification.

Quantifying the physisorbed CO2 via the FTIR band at 2345 cm -1 revealed a higher tendency of Ru(bpy)3-FAU towards CO2 adsorption than FAU (Figure S4). More specifically, an increase in (50 µg/mL). This observation was performed using blue excitation filter which covers excitation wavelength in the range of 420 -495nm and emission wavelength at 535 nm. That set of filter and excitation maxima of Ru(bpy)3-FAU (450nm) allows us to obtain green signal. After 72 h, a significant amount of Ru(bpy)3-FAU zeolite nanoparticles was predominantly localized around the nucleus and at much lesser extent, on the nucleus (Figure 6D). The orthogonal view was obtained following z-stacking of the region which was marked with red-dashed circle. Figure (6E) revealed that a small amount of particles entered into the nucleus. Our results are in line with the previous study demonstrating that small-nanoparticles (25 nm)

show higher nuclear delivery to gain nuclear entry in a passive manner. [START_REF] Tammam | The Effect of Nanoparticle Size and NLS Density on Nuclear Targeting in Cancer and Normal Cells; Impaired Nuclear Import and Aberrant Nanoparticle Intracellular Trafficking in Glioma[END_REF] In addition to that, the shape and size of particles was shown to have an influence on the uptake process by the cells. [START_REF] Cho | The Effects of Size, Shape, and Surface Functional Group of Gold Nanostructures on Their Adsorption and Internalization by Cells[END_REF][START_REF] Zhang | Size-Dependent Endocytosis of Nanoparticles[END_REF] The optimal nanoparticle radius for endocytosis was previously determined to be in the range of 25-30 nm, [START_REF] Zhang | Size-Dependent Endocytosis of Nanoparticles[END_REF] which is in a good agreement with prior estimates. [START_REF] Gao | Mechanics of Receptor-Mediated Endocytosis[END_REF] The current results are also in an agreement with other studies reported that nanoparticles with a size of 50 nm and 120 nm are internalized faster in comparison to 250 nm particles. [START_REF] Suen | Size-Dependent Internalisation of Folate-Decorated Nanoparticles via the Pathways of Clathrin and Caveolae-Mediated Endocytosis in ARPE-19 Cells[END_REF] In a very recent study, [START_REF] Vilaça | Internalization Studies on Zeolite Nanoparticles Using Human Cells[END_REF] the kinetics of uptake of zeolite L and NaY by Hs 578T breast cancer cells and MCF-10 epithelial mammary cells was presented. The NaY zeolite had cubic geometry with an average diameter of 700 nm, while zeolite L had disc-shaped particles with a size of around 400 nm. The cells started to engulf the NY particles with their filopodia, but only a few of them were inside the cell due to the big size and formation of aggregates. However, after 5 min of incubation more L zeolite particles were internalized than zeolite NaY.

It is also known that in addition to the size and shape, the surface charge of the particles have an impact on internalization efficacity. The surface functionalization of nanoparticles can be utilized to promote cellular uptake and determine nanoparticle sub-cellular localization. The most frequently postulated internalization pathway is endocytosis. However, the exact mechanism has been shown to be cell-type dependent. The cell nucleus intuitively becomes of primary interest for targeting nanoparticles and also has been proven to be the main interaction site for most therapeutic agents such as anticancer drugs, genes, free radicals, and heat.

Toxicity study of Ru(bpy)3-FAU zeolite nanoparticles

In vitro cytocompatibility of Ru(bpy)3-FAUzeolite nanoparticles on U251-MG cells was tested by crystal violet assay. Adherent cells detach from cell culture plates during the cell death. The difference in proliferation upon treatment with test agent is used for indirect quantification of cell death. In other words, cells that undergo cell death lose their adherence and are subsequently lost from the population of cells, reducing the amount of crystal violet staining in a culture.

U251-MG cells were treated with increased concentrations of Ru(bpy)3-FAU zeolite nanoparticles (50,100, 250 and 500 µg/mL), and viability was followed at Days 1, 4 and 7 (Figure 7). The effects of Ru(bpy)3-FAU zeolite nanoparticles on the cell growth was also shown by light microscopy images (Figure 7A). The density of the cells were relatively similar after 1 day of exposure. However, the number of the cells has decreased upon exposure to 50 and 100 µg/mL Ru(bpy)3-FAU zeolite nanoparticles after 4 days. Although the cells started to recover from the inhibitory effect of Ru(bpy)3-FAU and continue to proliferate along 7 days, concentration dependent slight decrease was observed. To further confirm these observations, the viability of the cells was quantified by measuring optical density following crystal violet staining (Figure 7B). After one day of exposure, in the range of the tested concentrations, the zeolite nanoparticles did not exhibit toxicity to the cells as confirmed by the stability (even a slight increase) in optical density. Ru(bpy)3-FAU showed concentration dependent decrease in the viability of U251-MG cells at Days 4 and 7 in a concentration dependent manner. However, the lethal dose 50% was not achieved even working with the highest concentration of Ru(bpy)3-FAU nanoparticles (500µg/ml) We can conclude that the Ru(bpy)3-FAU zeolite nanoparticles are safe for these cells. While using silver-containing zeolites even with low concentrations (as low as 50µg/ml) 3 no cells survived after 24 or 48 exposure was registered. 3

Conclusions

We successfully incorporated ruthenium-tris(2,2′-bipyridyl) complex within FAU zeolite nanocrystals. The FAU nanocrystals were loaded with 8 % ruthenium-tris(2,2′-bipyridyl). The introduction of this complex in the zeolite crystals did not change the morphology, crystallinity, and colloidal stability of the sample.

The ruthenium-tris(2,2′-bipyridyl) complex introduced in the FAU zeolite retained its absorption properties. Upon excitation at the MLCT band at 450 nm, an orange-red emission appeared at 628 nm, typical for organometallic complexes bearing π-conjugated ligands which is not common for pure zeolite.

The viability tests were performed reveling the concentration and time dependent toxicity of the Ru(bpy)3-FAU zeolite on U251 cells. The safest dose range of the Ru(bpy)3-FAU nanoparticles was in the range of 50-100 µg/mL. Furthermore, confocal microscopy imaging show the internalization of the zeolite nanocrystals in the cells owing to their luminescent properties inherited from the ruthenium complex. The Ru(bpy)3-FAU was shown to be able of crossing the cell membrane barrier and localized mostly at the perinuclear regions with some intranuclear uptake.

Being stable, safe, capable of loading various guest molecules (drugs ang gases) and instantaneously localized within the cells, the Ru(bpy)3-FAU nanosized zeolite crystals could be considered as a potential candidate for medical application.

Figure 1 .

 1 Figure 1. XRD patterns of (a) as-synthesized FAU and (b) Ru(bpy)3-FAU zeolite samples.

Figure 2 .

 2 Figure 2. (A) Particle size distribution and (B) zeta potential curves of (a) as-synthesized FAU

35

 35

Figure 3 .

 3 Figure 3. (A) Thermogravimetric (TG) analyses and (B) Differential thermogravimetric (dTG)

Figure 4 .

 4 Figure 4. Nitrogen adsorption/desorption isotherms of (a) as-prepared FAU and (b) Ru(bpy)3-

Figure 5 .

 5 Figure 5. (A) UV-vis absorption spectrum, (B) excitation (dotted line) and emission (continuous

Figure 6 .

 6 Figure 6. Epifluorescent (A, B, C) and confocal microscopy images (D, E) showing the uptake of Ru(bpy)3-FAU zeolite by U-251-MG cells. (A) (B) and (C) represent progressive uptake of the tracer molecule. (D) The top and magnified orthogonal view (E) of the U251-MG cells. Yellow and red arrows show cytoplasmic and intranuclear deposition of Ru(bpy)3-FAU, respectively.

Figure 7 .

 7 Figure 7. (A) Light microscopy images of U251-MG cells after crystal violet staining after 1 day, 4 and 7 days exposure to Ru(bpy)3-FAU zeolite. (B) The viability of human glioblastoma cells after 1 day, 4 and 7 days exposure to Ru(bpy)3-FAU zeolite samples with different concentrations (50,100, 250 and 500 µg/mL).

Table 1 .

 1 Surface area and pore volume of as-prepared FAU and the Ru(bpy)3-FAU samples determined by N2 sorption measurements.

	(a)
	(b)

Acknowledgement

The financial support provided by ZEOXY project (Conseil Régional de Basse Normandie) and the European Union-Fonds Européen de Développement Régional (FEDER) is acknowledged.

The authors thank the Institut National du Cancer (INCA-11699).

CO2 loading capacity of about 17 % was recorded for the Ru(bpy)3-FAU compared to the as prepared FAU zeolite. Despite its higher CO2 loading capacity, the adsorption on Ru(bpy)3-FAU was slower than on the as-prepared FAU zeolite, this is exemplified by the lower slope of the curve representing the change of the FTIR band area versus the applied CO2 pressure. This phenomenon is attributed to the partial obstruction of pores by the presence of ruthenium complex. The modified zeolite is capable of loading molecular oxygen, exemplified by the appearance of the band at 1552 cm -1 following the O2 adsoption experiment (Figure S5). The O2 loading capacity of the Ru(bpy)3-FAU sample was lower than that of the as-prepared FAU sample.

Nanosized Ru(bpy)3-FAU zeolite as an intracellular localization tracer

To exploit the potential of Ru(bpy)3-FAU zeolite nanoparticles as an intracellular localization tracer or in the future as a vector for delivery of either gases or drugs, the intracellular localization in U251-MG cells was determined. The U251-MG cells incubated with 50 µg/mL of zeolite nanoparticles for 4 h, 24 h and 72 h were studied by epifluorescent and confocal microscopy (Figure 6). After 4h of incubation, the Ru(bpy)3-FAU zeolite nanocrystals are concentrated around the cells (Figure 6A). Upon increasing the incubation time, the particles started to cross the cell membrane and accumulate into the cytoplasm around the nucleus (Figure 6B and6C). In this examination the green excitation was used. This filter encompass an excitation wavelength 510-580 nm and emission wavelength 620 nm which matches with emission maximum of the Ru(bpy)3-FAU and naturally red color was obtained. The intracellular localization of the Ru(bpy)3-FAU was studied by confocal microscopy. Figure 6D and 6E are the confocal micrographs of U251-MG cells after 72 h of incubation with the Ru(bpy)3-FAU zeolite