Romain Dubourget

Aude Sangare

Hélène Geoffroy

Thierry Gallopin

Dr Armelle Rancillac
email: armelle.rancillac@college-de-france.fr

Aude Sangaré

Multiparametric characterization of neuronal subpopulations in the ventrolateral preoptic nucleus

Keywords: Unsupervised clustering of VLPO neurons Sleep-promoting neuron, serotonin, noradrenaline, VLPO, Ward's clustering, low-threshold calcium spike (LTS)

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

Introduction

Compared to the large number of cell groups involved in arousal, relatively few populations of neurons have been shown to turn off the arousal system. In the anterior hypothalamus, the ventrolateral preoptic nucleus (VLPO) is considered as the main brain region responsible for the inhibition of wake-promoting neurons during slow-wave sleep (SWS) [START_REF] Economo | Sleep as a problem of localization[END_REF][START_REF] Sherin | Activation of ventrolateral preoptic neurons during sleep[END_REF][START_REF] Saper | Sleep state switching[END_REF]. Indeed, retrograde transport experiments identified a set of GABAergic neurons in the VLPO, containing the peptide galanin, which innervate all of the major components of the ascending arousal systems [START_REF] Sherin | Innervation of histaminergic tuberomammillary neurons by GABAergic and galaninergic neurons in the ventrolateral preoptic nucleus of the rat[END_REF][START_REF] Saper | Hypothalamic regulation of sleep and circadian rhythms[END_REF].

Two main classes of neurons have already been characterized, ex vivo in the VLPO of rats. The largest neuronal type displays triangular-shape, expresses galanin and is endowed with the ability to generate a low-threshold calcium spike (LTS). Moreover, these neurons are inhibited by several arousal neurotransmitters such as noradrenaline (NA) [START_REF] Gallopin | Identication of sleep-promoting neurons in vitro[END_REF][START_REF] Matsuo | alpha 2-Adrenoceptor-mediated presynaptic modulation of GABAergic transmission in mechanically dissociated rat ventrolateral preoptic neurons[END_REF][START_REF] Moore | Direct activation of sleep-promoting VLPO neurons by volatile anesthetics contributes to anesthetic hypnosis[END_REF][START_REF] Liu | Propofol stimulates noradrenalin-inhibited neurons in the ventrolateral preoptic nucleus by reducing GABAergic inhibition[END_REF][START_REF] Mccarren | 2-Adrenergic Stimulation of the Ventrolateral Preoptic Nucleus Destabilizes the Anesthetic State[END_REF][START_REF] Varin | Glucose Induces Slow-Wave Sleep by Exciting the Sleep-Promoting Neurons in the Ventrolateral Preoptic Nucleus: A New Link between Sleep and Metabolism[END_REF][START_REF] Scharbarg | Astrocyte-derived adenosine is central to the hypnogenic effect of glucose[END_REF], suggesting that they are maintained silent during waking. Thus, they are named NA (-) neurons and considered as putative sleep-promoting neurons. Two intermingled subtypes of NA (-) neurons were later demonstrated to differ in their firing responses to serotonin (5-HT). Indeed, in contrast to other neurotransmitters that promote wakefulness, 5-HT was shown to inhibit (44%; Type-1) or to excite (56%; Type-2) NA (-) neurons, revealing two potential different neuronal subtypes of putative sleep-promoting neurons [START_REF] Gallopin | The endogenous somnogen adenosine excites a subset of sleep-promoting neurons via A2A receptors in the ventrolateral preoptic nucleus[END_REF][START_REF] Fort | Alternating vigilance states: New insights regarding neuronal networks and mechanisms[END_REF].

Albeit far less studied, VLPO neurons excited by NA (i.e. NA (+) neurons) have only been sparsely described at the present time. In rats, they display a fusiform/bipolar shape and were described as devoid of LTS [START_REF] Gallopin | Identication of sleep-promoting neurons in vitro[END_REF][START_REF] Matsuo | alpha 2-Adrenoceptor-mediated presynaptic modulation of GABAergic transmission in mechanically dissociated rat ventrolateral preoptic neurons[END_REF][START_REF] Liu | Histamine regulates activities of neurons in the ventrolateral preoptic nucleus[END_REF]). These neurons are supposed to correspond to local neurons and are considered as non associated with sleep promotion.

To describe the neuronal diversity within the VLPO, here we first performed an exhaustive electrophysiological characterization of the neurons. Unsupervised clustering analysis based on these electrophysiological features revealed 3 different main classes and 5 subclasses of VLPO neurons. We furthermore determined their pharmacological responses to bath application of NA and 5-HT and performed Neurolucida reconstructions of the biocytin-filled VLPO neurons to reveal distinctive physiological and morphological properties among these neuronal subtypes. Altogether, our results establish the existence of a complex neuronal diversity within the VLPO that represents an important step toward understanding sleep regulation.

Materials and Methods

Animals.99 Male-only C57BL/6J mice (14-21 days old; Charles River, France) were used for all experiments. Mice arrived in the laboratory at least 1 week before experiments to acclimate to their new environment, and were housed in a temperature-controlled (20-22°C) room under a 12-hour light-dark cycle (lights on at 09:00 a.m.) with ad libitum access to food and water.

All animal procedures were conducted in strict compliance with our institutional protocols and were approved by the European Community Council Directive of 22 September 2010 (010/63/UE) and the local ethics committee (Comité d'éthique en matière d'expérimentation animale number 59, C2EA-59, 'Paris Centre et Sud'). The number of animals in our study was accordingly kept to the necessary minimum.

Preparation of acute hypothalamic slices. Animals were decapitated at the beginning of the light phase, between 09:00 and 10:00 a.m. Brains were quickly extracted without removing the meninges and submerged in cold slicing artificial cerebrospinal fluid (aCSF, 4°C) containing (in mM): 130 NaCl; 5 KCl;2.4 CaCl2;20 NaHCO3;1.25 KH2PO4;1.3 MgSO4; 15 sucrose; and 1 kynurenic acid (pH = 7.35). Brains were constantly oxygenated with 95% O2-5% CO2. Coronal brain slices (300 µm thick) containing the VLPO were cut with a vibratome (VT2000S; Leica) and transferred to a constantly oxygenated (95% O2-5% CO2) holding chamber containing aCSF. Subsequently, individual slices were placed in a submerged recording chamber maintained at 32°C and perfused with oxygenated kynurenate-free aCSF, and placed under an upright microscope (Zeiss) equipped with a CCD camera (Hamamatsu) for observation.

Drugs. Noradrenaline (NA, 50 µM; Sigma) and serotonin (5-HT, 100 µM; Sigma) were used in this study.

Whole-cell patch-clamp recordings. Slices were maintained immersed and continuously surperfused at 1-2 mL/min with oxygenated kynurinic acid-free aCSF. Electrophysiological experiments were performed with a MultiClamp700B (Axon Instruments) amplifier connected to an acquisition board (Digidata 1440; Axon Instruments) attached to a computer running the pCLAMP software (Axon Instruments). The targeted neurons were located in the VLPO.

Recordings in the whole-cell patch-clamp configuration were performed with patch-clamp pipettes (3-6 MΩ) filled with an internal solution containing (in mM): 144 K-gluconate; 1 MgCl2; 0.5 EGTA; 10 HEPES (pH 7.2); and 285-295 mOsm for 126 recorded neurons or with an internal solution containing (in mM): 144 K-gluconate; 3 MgCl2; 0.2 EGTA; 10 HEPESfor 163 recorded neurons. Electrophysiological features of these two populations were compared among all neurons and within each cluster, and no statistical differences were found.

For morphological analysis, 2 mg/mL biocytin (Sigma Aldrich) was added to these internal solutions. The liquid junction potential of the patch pipette and the perfused extracellular solution were 11 mV and were not applied to the data. The pipette was slowly brought to the selected neuron for recording in whole-cell configuration using infrared videomicroscopy guidance and clamped at -60 mV. The intrinsic neuronal membrane properties were assessed in current-clamp by applying current steps (800 ms) from -100 pA until firing saturation, in 10 pA increments.

For each neuron, 39 electrophysiological parameters were measured according to the Petilla terminology [START_REF] Ascoli | Petilla terminology: nomenclature of features of GABAergic interneurons of the cerebral cortex[END_REF], as fully described in [START_REF] Karagiannis | Classification of NPY-expressing neocortical interneurons[END_REF]. Briefly, (1) the resting membrane potential was measured immediately after passing to whole-cell configuration. (2) Input resistance (Rm) and (3) the membrane time constant (τm) were determined at the beginning of the voltage response of a hyperpolarization current step (10 pA, 800 ms). The time constant was determined by fitting this voltage response to a single exponential. (4) The membrane capacitance (Cm) was calculated according to the equation Cm = τm/Rm. Under our conditions, injection of hyperpolarizing current pulses often induced a hyperpolarization-activated cationic current (Ih) that followed the initial hyperpolarization peak, known as a sag. (5) Ghyp was measured as the slope of the linear portion of an I-V plot, measured at the beginning (0-0.1 s; -100 to 0 pA). ( 6) Gsag was measured as the slope of the linear portion of a current-voltage (I-V) plot, measured at the end of the hyperpolarizing current pulses (0.7-0.8 s; -100 to 0 pA). ( 7) ∆GSag corresponds to (Gsag-Ghyp)/Gsag. ( 8 The first spike latency was computed as the time needed to elicit a spike after the onset of a current pulse corresponding to the rheobase. Neurons have been described to exhibit a wide range of firing behaviors around threshold, some exhibiting bursting, adapting, regular or irregular trains of action potentials. In order to describe this variety of behaviors with quantitative parameters, the interspaced intervals measured in response to the minimal current injection eliciting more than three action potentials were plotted and fitted to a linear curve.

(11) Adaptation (mthreshold) was computed as the slope of the linear fit and (12) the minimal steady state frequency (Fthreshold). (13) The spontaneous discharge was measured in the cellattached configuration, just before the patch break down. When depolarized by current injection, VLPO neurons displayed several action potential discharged patterns. Initial burst of action potentials followed by a shorter 'interspike interval' than the steady-state trains of spikes was referred as ( 14) burst firing neurons. We also observed (15) non-adapting non-fast spiking firing pattern, (16) adapting firing, (17) late spiking firing and (18) irregular firing. (19 and 23) Amplitudes of the first (A1) and second (A2) action potential (AP) were measured from the threshold to the positive peak on the first step, where at least 3 APs were induced. (20 and 24) AP durations were measured at half-amplitude (D1 and D2). ( 21) The amplitude (AHPmax amplitude) and ( 22) duration of the maximal after-hyperpolarization (AHPmax) were measured for the first AP. (25) Amplitude variation (Var A) and (26) duration variation (Var D) were computed as (A1-A2)/A1*100 and (D2-D1)/D1*100 respectively. At higher stimulation intensities, the maximal firing rate was defined as the last trace before prominent reduction of the action potential amplitude, indicative of a saturated discharge. To take into account the biphasic spike frequency adaptation (early and late), instantaneous firing frequency was fitted to a single exponential with a sloping baseline, according to the equation Fsat = Asat × E -t /τsat + t × msat + Fmax, where (27) Asat corresponds to the amplitude of early frequency adaptation, (28) τsat to the time constant of early adaptation, (29) Fmax the maximal steady-state frequency and (30) msat to the slope of late adaptation. LTS VLPO neurons are believed to be the putative sleep-promoting. In addition to the Petilla terminology, current pulses of increased amplitude were applied on neurons hold at < -80 mV to determine the presence or absence of an (31) LTS. (32) LTS amplitude and (33) duration were also measured on the first step that elicited an LTS. (34) The number of AP elicited by the LTS (AP/burst) was counted and (35) the outward rectification in the positive direction (Slop) was measured.

(36) Occurrence of post-inhibitory rebound, (37) its amplitude, (38) duration and (39) the number of AP elicited by a post-inhibitory rebound following hyperpolarizing current of 100 pA from -60 mV were measured.

Pharmacological responses to NA and 5-HT applications were recorded in current-clamp mode.

Data were rejected if the access resistance increased over 25 MOhm during a recording or changed by more than 25% during the recording.

Ward's Clustering and Silhouette. Unsupervised clustering was performed essentially as described previously [START_REF] Karagiannis | Classification of NPY-expressing neocortical interneurons[END_REF][START_REF] Perrenoud | Characterization of Type I and Type II nNOS-Expressing Interneurons in the Barrel Cortex of Mouse[END_REF]. Briefly, 39 electrophysiological parameters were standardized by centering and reducing all of the values of the 289 neurons using Statistica 6 software (Statsoft). After the selection of significant principal components (principal component analysis with absolute loading values of 0.7), we conducted a cluster analysis using unsupervised clustering performed by Ward's method [START_REF] Ward | Hierarchical grouping to optimize an objective function[END_REF]) that does not require to set a number of classes to be characterized. The clusters generated by Ward's method were then corrected using the K-means algorithm by using Matlab software (MathWorks). To quantitatively assess the quality of a clustering, silhouette analysis of the Kmeans clustering was performed. A positive silhouette value indicates that on average, the neuron is closer to the neurons of its own cluster than from the neurons belonging to other clusters in the parameter space. On the opposite, a negative value indicates a potential misclassification. The 18 electrophysiological parameters used for clustering analysis were Rm

(2), τm (3), Cm (4), ∆GSag (7), first spike latency (10), mthreshold (11), Fthreshold ( 12 Morphological analysis. In order to describe variations of morphology between recorded neurons, a total of 40 parameters related to features of the soma, dendrites and axon were extracted. Morphologies of somata were reconstructed from infrared pictures taken prior to whole cell recording, while in the cell attached configuration with a CCD camera controlled by the Image-Pro 7 software (Media Cybernetics Inc.). Morphological features were measured from these images after calibration with a standard 24 µm grid. (40) The cell body area, (41) perimeter, (42) form factor, and (43) maximal, (44) minimal diameter passing through the centroid were computed. The degree of flatness of a contour shape, taken as the ratio of its minimum diameter to its maximum diameter, was indicated as (45) the aspect ratio. ( 46) The somatic solidity, the ratio of the somata area as a whole over the convex area, where values closer to 1 represent more solid (i.e. smooth, uniform) somata was calculated for each soma. The dendritic properties of recorded neurons were extracted by using Neurolucida explorer (MBF Bioscience) and Excel (Microsoft). In order to be able to describe the differences of dendritic arborizations, we systematically quantified the following parameters: (50) Number of primary dendrites, (51) total dendritic length, (52) total dendritic surface, (53) total dendritic volume, (54) ratio of the total dendritic length on the total dendritic surface, (55) highest dendritic order segment, (56) average tortuosity of dendritic segments (defined as the ratio of the length of the segment over the straight line path between its extremities) and ( 57) number of dendritic nodes. We performed a Sholl analysis [START_REF] Sholl | Dendritic organization in the neurons of the visual and motor cortices of the cat[END_REF]) to describe differences between neuronal arborization. (58) The length of the dendritic arborization that could be enclosed within a 100 μm radius circle around the cell body, (59) between 100 μm and 200 μm radii, (60) between 200 μm and 300 μm radii, and (61) outside a 300 μm radius was systematically extracted. Finally, (62-67) the number of dendritic spines in each segment order was extracted and (68) mean spine density was calculated.

Similarly, axonal properties were also extracted when the total axonal length of the reconstructed neurons exceeded 1 mm. ( 69) the percentage of axonal reconstruction per cluster was calculated. (70) Axonal length, (71) total axonal surface, (72) total axonal volume, (73) ratio of the total axonal length on the total axonal surface, (74) average tortuosity of axonal segments and (75) number of axonal nodes. We performed a Sholl analysis of digitized axonal morphology. Axonal length that could be enclosed within a 100 μm radius circle around the cell body, (76) between 100 μm and 200 μm radii, (77) between 200 μm and 300 μm radii, (78) between 300 μm and 400 μm radii, and (79) outside a 400 μm radius was systematically extracted.

Spontaneous excitatory and inhibitory currents. Spontaneous currents were recorded in voltage-clamp mode at a holding potential of -60 mV, which was less negative than the reversal potential for sIPSC. Indeed, for this set of experiments, we used the pipette solution contained a low concentration of Cl -(1 mM), resulting in an ECl of -111 mV. Therefore, inward currents were considered as sEPSCs and outward currents as sIPSCs. Data were filtered at 2 kHz, digitized at 10 kHz and acquired on-line using the pCLAMP 9 (Clampex) software (Axon Instruments). The recorded currents were subsequently analyzed using the Mini Analysis program (version 6.0.7; Synaposoft, Inc). The threshold for sIPSCs and sEPSCs detection was set at 7 pA, and the automatic detection was verified post hoc by visual inspection.

Statistics. All data are expressed as the mean ± standard error of the mean (SEM). Statistical analyses were performed using Statistica software (Statsoft). Differences between clusters were analyzed by one-way analysis of variance (ANOVA), followed by a Newman-Keuls multiple comparison test. Differences between two neuronal populations were analyzed using a Mann-Whitney U-test. P-values of ≤ 0.05 were considered statistically significant. In all cases, n refers to the number of examined neurons.

RESULTS

Unsupervised classification of VLPO neurons

Two hundred eighty nine neurons were recorded from acute preoptic hypothalamic slices in the VLPO. Neurons were first electrophysiologically characterized by patch-clamp recordings (see Materials and Methods). The spontaneous discharge was assessed in cell-attached configuration and the 38 others discriminative electrophysiological features were measured for each cell from responses to hyperpolarizing and depolarizing current pulses in whole-cell current-clamp mode (see Materials and Methods). 18 principal components were selected from a principal component analysis. Based on these parameters, we applied the unsupervised clustering Ward's method [START_REF] Ward | Hierarchical grouping to optimize an objective function[END_REF] to avoid a personal bias of determination and to group cells with large similarities into distinct subpopulations. A clustering tree was built, representing individual cells from its leaves and grouping them into the branched ramification up to a common root (Fig. 1A). The reliability of Ward's clustering was corrected using the K-mean algorithm. Two cells of cluster 1 were reassigned to cluster 3, and one cell of cluster 3 was reassigned to cluster 2 (Fig. 1B). In order to determine whether the allocation of neurons into three groups was the optimal outcome of the clustering process, our results were compared with clustering trials whereby thresholds were set to define fewer or higher numbers of groups. We established that a cluster analysis yielding three groups was the best outcome with a mean silhouette value of 0.275 (Fig. 1C). Similarly, we applied the clustering Ward's method on two separate groups of neurons (163 and 126 neurons respectively). These unsupervised classifications also determined 3 clusters endowed with the same proportion of neurons than observed in Figure 1A. 95.09% out of the 163 neurons and 92.06% out of the 126 neurons segregated in the same cluster than in Figure 1A. Thus, the subdivision of VLPO neurons into three main groups was retained for further analysis. However, it appeared from the dendrogram that neurons from cluster 2 and 3 were likely composed of 2 distinct subgroups that were named 2a, 2b and 3a, 3b

respectively. Therefore, we further analyzed the electrophysiological, morphological and pharmacological features of clusters 1, 2a, 2b, 3a and 3b to define their properties.

Electrophysiological diversity of VLPO neurons

To characterize the neuronal diversity of VLPO neurons, the 39 discriminative electrophysiological features measured on our 289 cells were compared among the five identified clusters. Electrophysiological data regarding subthreshold properties among VLPO neurons showed that cluster 3b neurons in particular display several distinctive features. They were characterized by a significantly higher input resistance (2) and an important Ih current, leading to higher slopes of I-V plots (5, 6) and a smaller sag index (7) compared to other groups (Table 1). Neurons from cluster 2b were also characterized by their highest membrane capacitance (4). When depolarized just above threshold, neurons from cluster 3b significantly exhibited a lower rheobase (9), whereas neurons from cluster 2b displayed significant longer first spike latency (10), compared to other neurons (Table 2). Neurons from cluster 1 were also distinguished from other groups by their higher adaptation (11) and steady state firing frequency (12) at threshold currents (Table 2). VLPO neurons also exhibited a wide diversity of evoked near threshold firing patterns (Fig. 2).

Burst firing neurons ( 14) were essentially found in cluster 1, non fast-spiking non-adapting firing neurons (15) in cluster 3b, adapting firing neurons (16) in cluster 2a, late spiking firing neurons (17) in cluster 2b and irregular firing neurons (18) in cluster 3a (Table 3). However, clusters 2a and 3a were also endowed with a high proportion of non fast-spiking non-adapting firing neurons. The presence of an LTS is one of the electrophysiological landmark of sleep-promoting neurons [START_REF] Gallopin | Identication of sleep-promoting neurons in vitro[END_REF][START_REF] Matsuo | alpha 2-Adrenoceptor-mediated presynaptic modulation of GABAergic transmission in mechanically dissociated rat ventrolateral preoptic neurons[END_REF][START_REF] Gallopin | Effect of the wake-promoting agent modafinil on sleep-promoting neurons from the ventrolateral preoptic nucleus: an in vitro pharmacologic study[END_REF][START_REF] Saint-Mleux | Nicotinic enhancement of the noradrenergic inhibition of sleep-promoting neurons in the ventrolateral preoptic area[END_REF][START_REF] Saint-Mleux | Suprachiasmatic modulation of noradrenaline release in the ventrolateral preoptic nucleus[END_REF][START_REF] Liu | Histamine regulates activities of neurons in the ventrolateral preoptic nucleus[END_REF][START_REF] Moore | Direct activation of sleep-promoting VLPO neurons by volatile anesthetics contributes to anesthetic hypnosis[END_REF]) that was frequently observed in clusters 1 and 3 (31; Table 5 and Fig. 2). However, we observed no statistical difference regarding LTS properties among VLPO clusters. An outward rectification (35) appeared to be a specific feature of cluster 2 neurons (Table 5). The post-inhibitory rebound following hyperpolarizing current from -60 mV was frequently recorded and also represents a discriminative parameter of VLPO neurons (Table 6). In particular, cluster 2 neurons were significantly less frequently endowed with a post-inhibitory rebound (36) than neurons from clusters 1 and 3. Post-inhibitory rebounds also had distinct kinetics among the VLPO neuronal clusters: mean amplitude (37) was significantly higher in cluster 1 vs. the other clusters, and lasted longer (38) in the cluster 3b (Table 6). Finally, we found that neurons from cluster 3b fired significantly more action potential per rebound than neurons from clusters 2a, 2b and 3a (39; Table 6).

Morphological properties of VLPO neurons

Basic morphometric analyses of somata were determined from infrared picture taken prior recording. Multipolar cells with round, polygonal and triangular somata as bipolar/fusiform neurons were found in relative equivalent proportion in all neuronal clusters. Yet, to avoid a subjective classification of somata shape, morphological parameters were extracted from their measurements. In particular, this analyze revealed that neurons from the cluster 2b displayed a cell body perimeter significantly bigger than somata from other clusters (41; Table 7).

(45) Aspect ratio 1.7 ± 0.1 1.6 ± 0 1.6 ± 0.1 1.7 ± 0.1 1.6 ± 0 - - - - - (46) Solidity 0.9 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 - - - - - (47) Convexity 1 ± 0 1 ± 0 1 ± 0 1 ± 0 1 ± 0 - - - - - (48) Roundness 1.4 ± 0 1.3 ± 0 1.3 ± 0 1.3 ± 0 1.3 ± 0 - - - - - (49) Compactness 1.2 ± 0 1.1 ± 0 1.2 ± 0 1.2 ± 0 1.1 ± 0 - - - - -
n, Number of cells; < indicates significantly smaller with P ≤ 0.05; << indicates significantly smaller with p ≤ 0.01; <<< indicates significantly smaller with P ≤ 0.001; one-way ANOVA, followed by a Newman-Keuls test. Values significantly different in one cluster vs. all others were highlighted in gray.

cases, axons were cut at the surface of the slices and were not possible to track (69, Table 9).

None of these axonal parameters are distinguish specifically in a cluster compared to others (Table 9). Nevertheless, we established that in 43% of the cases the axon arose directly from the soma, whereas in 37% of the reconstructed neurons the axon arose from a primary dendrite and in remaining 20% of the cases, the axon arose from the secondary or even from the tertiary dendrite. The mean distance of the axonal arising was at 12.94 ± 2.21 µm from the soma.

) Length/surface 1.0 ± 0.1 1.3 ± 0.1 1.0 ± 0.1 1.3 ± 0.1 1.1 ± 0.1 (µm -1 ) - - - - - (74) Tortuosity 0.6 ± 0.1 0.5 ± 0.1 0.6 ± 0.4 0.4 ± 0.0 0.6 ± 0.2 - - - - - (73

Spatial mapping of the distinct neuronal subpopulations into the VLPO

Reconstructed neurons were spatially mapped across three different brain sections of the VLPO, rostral to caudal (Fig. 4). The superimposed mapping of the neuronal subpopulations within the VLPO did not reveal a preferred location of any subpopulations that seem homogeneously spread. In the plane (A) are represented 6 neurons from cluster 1 (yellow, circle); 10 neurons from cluster 2a (light green, circle), 4 neurons from cluster 2b (dark green, star), 12 neurons from cluster 3a (light blue, circle) and 15 neurons from cluster 3b (dark blue, triangle). In the plane (B) are represented 6 neurons from cluster 1 (yellow); 11 neurons from cluster 2a (light green), 4 neurons from cluster 2b (dark green), 8 neurons from cluster 3a (light blue) and 7 neurons from cluster 3b (dark blue). In the plane (C), are represented 6 neurons from cluster 1 (yellow); 5 neurons from cluster 2a (light green), 3 neurons from cluster 2b (dark green), 2 neurons from cluster 3a (light blue) and 7 neurons from cluster 3b (dark blue). Insert: Coronal brain section showing the localization of the zoom presented in (A-C). Abbreviations: 3V, third ventricle; ox, optic chiasm.

Pharmacological properties of VLPO neurons

In the VLPO, sleep-promoting neurons are usually identified according to their inhibitory response to bath application of NA [START_REF] Gallopin | Identication of sleep-promoting neurons in vitro[END_REF][START_REF] Gallopin | The endogenous somnogen adenosine excites a subset of sleep-promoting neurons via A2A receptors in the ventrolateral preoptic nucleus[END_REF][START_REF] Moore | Direct activation of sleep-promoting VLPO neurons by volatile anesthetics contributes to anesthetic hypnosis[END_REF][START_REF] Liu | Propofol stimulates noradrenalin-inhibited neurons in the ventrolateral preoptic nucleus by reducing GABAergic inhibition[END_REF][START_REF] Varin | Glucose Induces Slow-Wave Sleep by Exciting the Sleep-Promoting Neurons in the Ventrolateral Preoptic Nucleus: A New Link between Sleep and Metabolism[END_REF]. Moreover, their response to 5-HT was also reported to segregate two subsets of VLPO sleep-promoting neurons [START_REF] Gallopin | The endogenous somnogen adenosine excites a subset of sleep-promoting neurons via A2A receptors in the ventrolateral preoptic nucleus[END_REF]. Therefore 189 and 132 neurons out of the 289 neurons of our data set were respectively tested for their response to NA (50 µM for 30 s) and to 5-HT (100 µM for 5 s) application. 111 of these neurons were tested for both neurotransmitters.

We found that on average 42% of VLPO neurons were hyperpolarized by NA application. The highest proportion of NA (-) neurons was observed in the cluster 3 (61%; n = 51), whereas neurons in cluster 1 and 2 were mainly NA (+) (60%, n = 18 and 76%, n = 55 respectively, Fig. 3B). To determine if NA (-) vs. NA (+) neurons displayed specific features, we compared their properties. Thus, we found in cell-attached recordings that NA (-) cells spontaneously discharged at higher firing rate than NA (+) cells (4.7 ± 0. Morphologically, NA (+) neurons were shown to have bigger somata than NA (-) neurons.

Their somatic perimeter was of 61.1 ± 1.2 vs. 55.9 ± 1.4 µm respectively (P < 0.039, Mann-Whitney U-test) with a higher mean dendritic length of 5922.3 ± 286.8 vs. 4425.5 ± 310.1 µm (P < 0.011, Mann-Whitney U-test). Here again, no correlation was found between the somatic shape and the neuronal response to NA. We found that only 41% of the triangular/multipolar cells in the VLPO were inhibited by NA, and conversely that 62% fusiform/bipolar cells excited by NA, confirming the previous observation than on the opposite to rat observations, somata of mice VLPO neurons are not predictive of their response to NA [START_REF] Wang | Morphine Inhibits Sleep-Promoting Neurons in the Ventrolateral Preoptic Area Via Mu Receptors and Induces Wakefulness in Rats[END_REF]).

In the 130 neurons tested for their response to 5-HT application, 72% were depolarized by the 5-HT. Cluster 2b presented the smallest proportion of 5-HT (+) neurons (50%; n = 16), close to the amount of 5-HT (+) neurons observed in cluster 3a (55%). Neurons from cluster 1, 2a and 3b included higher proportion of 5-HT (+) neurons, 84.2, 75 and 82.1% respectively.

Interestingly, the comparison of 5-HT (+) and 5-HT (-) neuronal features revealed the existence of only one discriminative parameter: the capacitance (58.3 ± 2.9 vs. 74.5 ± 6.9 pF respectively; P < 0.04, Mann-Whitney U-test).

In the 110 neurons both tested for their response to NA and 5-HT, we found 44% of NA (+)/5-HT (+) cells, 32% of NA (-)/5-HT (+) cells, 20% of NA (-)/5-HT (-) cells, and 12% of NA (+)/5-HT (-) cells. These four populations had similar electrophysiological and morphological properties, except for the NA (+)/5-HT (-) neurons that were characterized by a low input resistance of 491.4 ± 107.4 MΩ, high membrane capacitance of 100.0 ± 13.2 pF and ∆Gsag of 84.8 ± 3.3% and a long first spike delay of 220.8 ± 62.2 ms compared to the other groups.

To determine whether the inclusion of these pharmacological responses could provide discriminative parameter to define VLPO subclasses, we performed a Ward's clustering on these 111 neurons tested for both the NA and 5-HT effects. However, this approach provided a dendrogram with a smaller distance of aggregation and with a lower silhouette value. Therefore, these pharmacological properties were only kept as a read out in each identified clusters (Fig. 3B).

Spontaneous synaptic postsynaptic currents

Spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs) were recorded in 66 neurons out of the 289 cells of our database. However, due to a high intercellular variability, no significant differences were observed between the clusters, neither regarding the amplitude nor the frequency of both sEPSCs (from 18.97 ± 2.56 to 21.88 ± 1.33 pA; P > 0.611; and 0.91 ± 0.14 to 1.67 ± 0.44 Hz; P > 0.829, one-way ANOVA test) and sIPSCs (from 13.91 ± 2.47 to 15.46 ± 0.68 pA; P > 0.810; and 0.14 ± 0.1 to 0.79 ± 0.43 Hz; P > 0.294, one-way ANOVA; Fig. 6).

Discussion

Given the critical role of the VLPO in sleep induction and maintenance, the characterization of its neuronal populations is essential for a better understanding of their role in sleep regulation.

Using unsupervised clustering method on recorded neurons from acute slices of mice, we have defined for the first time the existence of five distinct cell subpopulations in the VLPO.

In this study, we recorded 289 neurons from the VLPO. 39 electrophysiological parameters were measured for each neuron. To define the existence of distinct neuronal subpopulations among VLPO neurons, we used the Ward's method which is usually used to group cells with large similarities and to avoid a personal bias of interpretation [START_REF] Cauli | Molecular and physiological diversity of cortical nonpyramidal cells[END_REF][START_REF] Karagiannis | Classification of NPY-expressing neocortical interneurons[END_REF]). Here, we performed a Ward's clustering on 18 electrophysiological parameters.

Interestingly, we found that intrinsic burst firing measured in cell-attached configuration was a strong discriminative feature. 19% of VLPO neurons were shown to display intrinsic burst. This result is in good agreement with a previous observation reporting 14% of burst firing neurons in the VLPO [START_REF] Morairty | Disinhibition of ventrolateral preoptic area sleep-active neurons by adenosine: a new mechanism for sleep promotion[END_REF]). These neurons segregated mainly in the cluster 1 (83%)

and to a lesser extent in the cluster 3 (17%). Neurons from cluster 1 were also characterized by their short lasting and low amplitude AHP compared to neurons from the other clusters.

Moreover, their second action potential evoked by a current pulse was significantly smaller.

Neurons from cluster 1 frequently displayed an LTS (80%) and 40% of these neurons were inhibited by NA application. These two hallmarks of sleep-promoting neurons suggest that at least some neurons from the cluster 1 could correspond to sleep-promoting neurons.

Cluster 2 VLPO neurons were composed of two distinct subsets: cluster 2a and cluster 2b cells, both exhibiting a high proportion of NA (+) cells (74 and 81.9% respectively) with a low occurrence of LTS (4.7 and 21.1% respectively). In particular, neurons from the cluster 2b strongly differentiated themselves from all other clusters. Pharmacologically, they were shown to contain the highest amount of 5-HT (-) neurons (50%). Electrophysiologically, Cluster 2b cells were characterized by a significantly higher membrane capacitance, a long delay to spiking and a strong outward rectification in the positive direction. Interestingly, these features were already reported to be the main characteristics distinguishing cholinergic basal forebrain neurons [START_REF] Khateb | Pharmacological and immunohistochemical evidence for serotonergic modulation of cholinergic nucleus basalis neurons[END_REF][START_REF] Unal | Adult mouse basal forebrain harbors two distinct cholinergic populations defined by their electrophysiology[END_REF]. These results suggest that on the opposite of rat where cholinergic neurons are confined into the basal forebrain, these neurons could be more sparsely segregated in mice brains. We speculate that neurons from cluster 2a could therefore correspond to local interneurons, whereas neurons from cluster 2b could correspond to cholinergic neurons.

Neurons from cluster 3 frequently exhibited an LTS (84%) and were in majority inhibited by NA application (61%). Interestingly, these neurons segregated into two different subpopulations, cluster 3a and 3b. Neurons from cluster 3b in particular were shown to have the smallest membrane resistance, the largest voltage sags, the lowest rheobase, the longer rebound duration, and numerous action potentials generated by their post-inhibitory rebound, compared to other VLPO neurons. In general, dendritic arborization of VLPO neurons were simple and quite similar among the clusters. Hence, neurons from cluster 3b displayed a particularly small somato dendritic tree. Finally, cluster 3b neurons also contained the highest proportion of 5-HT (+) neurons (82%).

To examine if synaptic inputs could differentiate VLPO neurons, we recorded sEPSCs and sIPSCS of 66 cells. However, we observed an important variation of sEPSCs and sIPSCS amplitude and frequency within each cluster. Thus, no statistical differences were found among VLPO clusters.

The specific features of VLPO neuronal clusters that we have defined in this study suggest that these different subpopulations could be involved in distinct aspects of sleep physiology. The role of some VLPO neurons in the thermoregulation being previously reported [START_REF] Lu | Effect of lesions of the ventrolateral preoptic nucleus on NREM and REM sleep[END_REF],

we speculate that one of these clusters could be involved in this function. Future studies will be needed to investigate the physiological roles of these 5 neuronal subpopulations.

Conclusions

In the present study, we identified 5 distinct neuronal subpopulations in the VLPO, characterized by specific electrophysiological, morphological and pharmacological properties that might be involved in specific functions of sleep regulation. These different subpopulations suggest a complex neuronal network organization within the VLPO.

  ) Action potential threshold corresponded to the voltage threshold of the first action potential elicited by a current ramp. (9) The rheobase was the minimum current that elicited an action potential.(10)

  ), bursting firing (14), late spiking (17), Var A (25), Var D (26), AHPmax amplitude (21), AHPmax duration (22), msat (30), LTS occurrence (31), rebound amplitude (37), rebound duration (38), and number of AP elicited by rebound (39).

( 47 )

 47 Convexity = (Convex Perimeter)/(Perimeter). This parameter is indicative of the somatic morphological profile. The measurement of how closely this shape approached that of a π) × area]/(feret max); numerical values of somatic compactness closer to 1 represent a more compact soma.

Fig. 1 .

 1 Fig. 1. Unsupervised clustering of VLPO neurons based on their electrophysiological properties. A, Ward's clustering of 289 VLPO neurons. Individual cells are represented along the x-axis. The y-axis represents the average within-cluster linkage distance in a space of 18 electrophysiological variables. Three clusters, 1 (in yellow), 2 (in green) and 3 (in blue) were identified. B, Clusters generated by Ward's method in (A) were corrected using the clustering output generated by the K-means algorithm. C, The silhouette analysis was used to assess the

Fig. 2 .

 2 Fig. 2. Electrophysiological properties of VLPO neurons recorded in current-clamp. A, Typical electrophysiological behavior of neurons from each cluster. Neurons from cluster 1 (yellow), 2a (light green), 2b (dark green), 3a (light blue) and 3b (dark blue) were recorded using wholecell patch-clamp recordings in response to the application of current pulses (bottom traces). Strong depolarizing current (top trace) evoked a high and sustained firing in all clusters. Top insets, IR pictures of the recorded neurons (scale bar, 20 µm). B, Characterization of LTS and

Fig. 4 .

 4 Fig. 4. Schematic distribution of the biocytin-labeled neurons within the VLPO. A, B and C represent three different planes at ~0.2 mm intervals rostral to caudal, around Bregma 0 mm, where VLPO neurons were recorded. The five subpopulations of VLPO neurons are color coded. In the plane (A) are represented 6 neurons from cluster 1 (yellow, circle); 10 neurons

Fig. 6 .

 6 Fig. 6. Recording of spontaneous excitatory and inhibitory synaptic inputs to VLPO neurons. A, The amplitude of both sEPSCs and sIPSCs revealed no statistical differences among the 5 clusters. B, The frequency of both sEPSCs (left panel) and sIPSCs (right panel) revealed no statistical differences among the 5 clusters.

Table 1 . Comparison of passive membrane properties among VLPO neuron subtypes

 1

		Cluster 1	Cluster 2a	Cluster 2b	Cluster 3a	Cluster 3b
		(n = 46)	(n = 85)	(n = 38)	(n = 52)	(n = 68)
	(1) RMP (mV)	-40.0 ± 1.1	-44.2 ± 0.7	-41.9 ± 0.7	-42.7 ± 0.7	-42.2 ± 0.6
		-	-	-	-	-
	(2) Rm (M)	557.9 ± 42.1	585.1 ± 29.8	626.5 ± 38.7	626.9 ± 24.1	991.3 ± 42.5
		1<<<3b	2a<<<3b	2b<<<3b	3a<<<3b	3b>>>1,2a,2b,3a
	(3) τm (ms)	32.9 ± 2.5	31.3 ± 2.1	41.6 ± 1.8	27.9 ± 1.3	41.4 ± 1.7
		1<<2b	2a<<<2b,3b	2b>1>>>2a,3a	3a<<<2b,3b	3b>>>2a,3a
	(4) Cm (pF)	63.6 ± 3.7	56.7 ± 2.5	86.3 ± 4.4	47.4 ± 2.0	45.7 ± 1.7
		3b<<3a<1<<<2b	2a<<<2b	2b>>>>1,2a,3a,3b	3a<1<<<2b	3b<<1<<<2b
	(5) Ghyp (M)	389.7 ± 30.7	383.0 ± 17.7	480.5 ± 28.8	444.7 ± 17.0	700.4 ± 25.7
		1<<<3b	2a<<<3b	2b<<<3b	3a<<<3b	3b>>>1,2a,2b,3a
	(6) Gsag (M)	290.3 ± 21.5	311.6 ± 13.4	359.1 ± 17.3	362.2 ± 12.2	427.5 ± 16.2
		1<<<3b	2a<<<3b	2b<3b	3b>3a	3b>2b,3a>>>1,2a
	(7) ∆Gsag (%)	76.8 ± 2.1	83.8 ± 1.2	80.4 ± 1.3	82.7 ± 0.8	63.5 ± 1.6
		3b<<<1<2a	2a>1>>>3b	2b>>>3b	3a>>>3b	3b<<<1,2a,2b,3a

n, Number of cells; < indicates significantly smaller with P ≤ 0.05; << indicates significantly smaller with P ≤ 0.01; <<< indicates significantly smaller with P ≤ 0.001; one-way ANOVA, followed by a Newman-Keuls test. Values significantly different in one cluster vs. all others were highlighted in gray.

Table 2 . Comparison of just above threshold properties among VLPO neuron subtypes

 2

		Cluster 1	Cluster 2a	Cluster 2b	Cluster 3a	Cluster 3b
		(n = 46)	(n = 85)	(n = 38)	(n = 52)	(n = 68)
	(8) APth (mV)	-40.5 ± 0.7	-39.8 ± 0.4	-40.1 ± 0.6	-42.4 ± 0.5	-43.7 ± 0.4
		-	-	-	-	-
	(9) Rheobase (pA)	22.4 ± 2.0	30.0 ± 1.5	24.9 ± 2.0	18.5 ± 1.0	11.9 ± 0.5
		3b<<<1<2a	2a>1>>>3a,3b	3b<<<2b	3b<3a<<<2a	3b<3a<<<1,2a,2b
	(10) 1 st spike (ms)	96.2 ± 7.9	113.6 ± 5.7	287.4 ± 17.5	88.7 ± 5.1	86.8 ± 5.7
		1<<<2b	2a<<<2b	2b>>>1,2a,3a,3b	3a<<<2b	3b<<<2b
	(11) mth (Hz/s)	-274.0 ± 105.0	-9.2 ± 2.2	-5.8 ± 1.8	-11.7 ± 1.6	-8.5 ± 1.2
		1<<<2a,2b,3a,3b	2a<<<1	2b<<<1	3a<<<1	3b<<<1
	(12) Fth (Hz)	50.2 ± 4.6	7.6 ± 0.4	8.2 ± 0.7	11.0 ± 0.6	12.2 ± 11.0
		1>>>2a,2b,3a,3b	2a<<<1	2b<<<1	3a<<<1	3b<<<1

n, Number of cells; < indicates significantly smaller with P ≤ 0.05; << indicates significantly smaller with P ≤ 0.01; <<< indicates significantly smaller with P ≤ 0.001; one-way ANOVA, followed by a Newman-Keuls test. Values significantly different in one cluster vs. all others were highlighted in gray.

Table 3 . Comparison of firing properties among VLPO neuronal subtypes

 3

		Cluster 1	Cluster 2a	Cluster 2b	Cluster 3a	Cluster 3b
		(n = 46)	(n = 85)	(n = 38)	(n = 52)	(n = 68)
	(13) Spont. (Hz)	3.6 ± 0.7	1.9 ± 0.3	2.7 ± 0.2	3.8 ± 0.3	4.6 ± 0.3
		-	2a<3b	2b<3b	-	3b>2a,2b
	(14) Burst (%)	97.8	0	0	11.5	4.4
		1>>>2a,2b,3a,3b	2a<<<1	2b<<<1	3a<<<1	3b<<<1
	(15) Non fast spiking	0	51.8	2.6	48.1	86.8
	non adapting (%)	1<<<2a,3a,3b	3b>>>2a>>>1,2b	2b<<<2a,3a,3b	1,2b<<<3a<<<3b	3b>>>1,2a,2b,3a
	(16) Adpating (%)	0	40.0	0	23.1	4.4
		1<<3a<<<2a,3b	2a>3a>>>1,2b,3b	1<<2b<<<2a,3b	2a>3a>>1,3a,3b	3b<<3a<<<2a
	(17) Late spiking (%)	2.2	0	97.4	0	1.5
		1<<<2b	2a<<<2b	2b>>>1,2a,3a,3b	3a<<<2b	3b<<<2b
	(18) Irregular (%)	0	8.2	0	17.3	2.9
		1<<<3a	2a<3a	2b<<3a	3a>2a>>2b,3b>>>	3b<<3a
					1
	n, Number of cells; < indicates significantly smaller with P ≤ 0.05; << indicates significantly
	smaller with P ≤ 0.01; <<< indicates significantly smaller with P ≤ 0.001; one-way ANOVA
	followed by a Newman-Keuls test . Values significantly different in one cluster vs. all others
	were highlighted in gray.
	Comparison of action potential properties among VLPO neurons essentially revealed that
	neurons from cluster 1 exhibit significant smaller AHP amplitude (21) and duration (22), a
	reduced second action potential amplitude (23, Table 4). Accordingly, they displayed a higher

Table 4 . Comparison of action potential properties among VLPO neurons

 4

		Cluster 1	Cluster 2a	Cluster 2 b	Cluster 3a	Cluster 3 b
		(n = 46)	(n = 85)	(n = 38)	(n = 52)	(n = 68)
	(19) A1 (mV)	89.3 ± 2.6	91.0 ± 1.5	91.8 ± 1.2	97.4 ± 1.8	90.5 ± 1.2
		-	-	-	-	-
	(20) D1 (ms)	1.1 ± 0.0	1.1 ± 0.0	1.2 ± 0.0	1.1 ± 0.0	1.2 ± 0.0
		-	-	-	-	-
	(21) AHPmax	-1.4 ± 0.3	-10.1 ± 0.4	-10.2 ± 0.6	-5.2 ± 0.3	-8.9 ± 0.4
	amplitude (mV)	1<<<2a,2b,3a,3b	2a>>>1,3a	2b>>>1,3a	2b,3b>>>3a>>>1	3b>>>1,3a
	(22) AHPmax	6.1 ± 3.1	131.9 ± 5.5	130.7 ± 5.8	99.5 ± 6.7	90.8 ± 5.5
	duration (ms)	1<<<2a,2b,3a,3b	2a>>>1,3a,3b	2b>>>1,3a,3b	2a,2b>>>3a>>>1	2a,2b>>>3b>>>1
	(23) A2 (mV)	69.8 ± 2.6	85.3 ± 1.3	88.1 ± 1.3	89.3 ± 1.8	85.1 ± 1.1
		1<<<2a,2b,3a,3b	2a>>>1	2b>>>1	3a>>>1	3b>>>1
	(24) D2 (ms)	1.4 ± 0.1	1.2 ± 0.0	1.2 ± 0.0	1.2 ± 0.0	1.3 ± 0.0
		1>2a,3a	2a<1	-	3a<1	-
	(25) Var A (%)	22.0 ± 1.8	6.1 ± 0.4	3.9 ± 0.5	8.4 ± 0.6	6.0 ± 0.4
		1>>>2a,2b,3a,3b	2a>>>1	3b>>>1	3a>>>1	3b>>>1
	(26) Var D (%)	-24.5 ± 3.8	-7.0 ± 0.6	-5.9 ± 0.5	-8.4 ± 0.8	-6.2 ± 0.6
		1>>>2a,2b,3a,3b	2a<<<1	2b<<<1	3a<<<1	3b<<<1
	(27) Asat (Hz)	92.2 ± 5.9	62.2 ± 5.0	39.0 ± 4.3	61.9 ± 2.3	54.5 ± 3.1
		1>>>2a,2b,3a,3b	2b<2a<<<1	2b<2a3a<<<1	2b<3a<<<1	3b<<<1
	(28 ) τsat (ms)	26.0 ± 2.6	43.2 ± 6.2	37.3 ± 4.6	28.0 ± 1.5	51.7 ± 7.1
		-	-	-	-	-
	(29) Fmax (Hz)	44.8 ± 5.2	48.0 ± 2.6	59.9 ± 5.6	48.8 ± 2.9	58.9 ± 3.2
		-	-	-	-	-
	(30) msat (Hz/s)	-18.9 ± 2.2	-15.6 ± 1.70	-22.4 ± 2.6	-5.6 ± 5.9	-17.2 ± 1.3
		-	-	-	-	-

n, Number of cells; < indicates significantly smaller with P ≤ 0.05; << indicates significantly smaller with P ≤ 0.01; <<< indicates significantly smaller with P ≤ 0.001; one-way ANOVA followed by a Newman-Keuls test . Values significantly different in one cluster vs. all others were highlighted in gray.

Table 5 . Comparison of LTS properties among VLPO neurons

 5

		Cluster 1	Cluster 2a	Cluster 2b	Cluster 3a	Cluster 3b
		(n = 37)	(n = 4)	(n = 8)	(n = 47)	(n = 54)
	(31) LTS (%)	80.4	4.7	21.1	90.4	79.4
		1>>>2a,2b	2a<<2b<<<1,3a,3b	2a<<2b<<<1,3a,3b	3a>>>2a,2b	3b>>>2a,2b
	(32) LTS	19.7 ± 1.3	13.8 ± 0.4	11.0 ± 2.7	15.8 ± 1.1	14.8 ± 1.0
	amplitude (mV)	-	-	-	-	-
	(33) LTS	178.6 ± 14.9	200.8 ± 37.8	213.9 ± 48.5	187.0 ± 12.1	225.9 ± 21.0
	duration (ms)	-	-	-	-	-
	(34) AP/burst	3.1 ± 0.4	1.2 ± 0.0	1.5 ± 0.3	1.9 ± 0.2	2.4 ± 0.2
		-	-	-	-	-
	(35) Slope (%)	0.2 ± 0.2	1.3 ± 0.4	3.7 ± 1.5	0.0 ± 0.0	0.0 ± 0.0
		1<<<2b	2a<2b	2b>2a>>>1,3a,3b	3a<<<2b	3b<<<2b
	n, Number of cells; < indicates significantly smaller with P ≤ 0.05; << indicates significantly

smaller with P ≤ 0.01; <<< indicates significantly smaller with P ≤ 0.001; one-way ANOVA, followed by a Newman-Keuls test. Values significantly different in one cluster vs. all others were highlighted in gray.

Table 6 . Comparison of overshoot of the membrane potential properties among VLPO neurons

 6 Number of cells; < indicates significantly smaller with P ≤ 0.05; << indicates significantly smaller with P ≤ 0.01; <<< indicates significantly smaller with P ≤ 0.001; one-way ANOVA, followed by a Newman-Keuls test. Values significantly different in one cluster vs. all others were highlighted in gray.

		Cluster 1	Cluster 2a	Cluster 2b	Cluster 3a	Cluster 3b
		(n = 37)	(n = 15)	(n = 19)	(n = 42)	(n = 66)
	(36) Rebound (%)	80.4	17.7	50.0	80.8	97.1
		3b>>>1>>>2a,2b	2a<<<1,2b,3a,3b	2a<<<2b<<<1,3a,3b	3b>3a>>>2a,2b	3b>3a>>>1,2a,2b
	(37) Rebound	22.5 ± 1.2	12.5 ± 0.5	11.8 ± 0.5	17.4 ± 0.7	16.0 ± 0.6
	amplitude (mV)	1>3a>>3b>>>2a,2 b	2a<3a<<<1	2b<3a<<<1	2a,2b<3a<1	3b<<1
	(38) Rebound	501.6 ± 64.8	264.0 ± 27.0	693.6 ± 37.5	365.0 ± 24.2	1022.1 ± 45.4
	duration (ms)	3b>>>1>>2a	2a<<1,2b<<<3b	3b>>2b>2a>>3a	3b>>>2b>3a	3b>>2b>>>1,2a,3a
	(39) AP/burst	4.1 ± 0.50	2.0 ± 0.2	3.0 ± 0.2	2.4 ± 0.1	6.4 ± 0.7
		1>>2a,3a	2a<<3b	3b>2b	3a<3b	3b>2b,3a>>2a

Table 7 . Somatic properties among VLPO neurons

 7

		Cluster 1	Cluster 2a	Cluster 2b	Cluster 3a	Cluster 3b
		(n = 46)	(n = 85)	(n = 38)	(n = 52)	(n = 68)
	(40) Cell body area (µm²)	174.6 ± 8.7	192.9 ± 8.4	232 ± 11.4	173.6 ± 7.7	205.9 ± 9.6
		1<<<2b	2<<2b	2b>>2a>>>1,3a	3a<<<2b	-
	(41) Perimeter (µm)	54.4 ± 1.9	54.8 ± 1.4	61.4 ± 1.7	53.4 ± 1.4	56.4 ± 1.4
		1<<2b	2a<<2b	2b>3b>>1,2a,3a	3a<<2b	3b<2b
	(42) Form factor	0.8 ± 0	0.8 ± 0	0.8 ± 0	0.8 ± 0	0.8 ± 0
		-	-	-	-	-
	(43) Feret max (µm)	20.5 ± 0.8	20.6 ± 0.6	22.9 ± 0.8	20.4 ± 0.6	21.1 ± 0.6
		-	-	-	-	-
	(44) Feret min (µm)	12.4 ± 0.4	12.9 ± 0.3	14.5 ± 0.5	12.3 ± 0.3	13.6 ± 0.4
		1<3b<<<2b	2a<2b	2b>2a>>>1,3a	<<<2b	-

Table 9 . Axonal properties among VLPO neurons

 9

			Cluster 1	Cluster 2a	Cluster 2b	Cluster 3a	Cluster 3b
			(n = 9)	(n = 10)	(n = 5)	(n = 7)	(n =10)
	(69) Axon > 1000 µm (%)	64.3	62.5	55.6	41.2	43.5
			-	-	-	-	-
	(70) Length (µm)	3377.9 ± 383.9 2665.7 ± 314.4 3336.1 ± 865.4 2808.1 ± 452.4 2438.0 ± 290.4
			-	-	-	-	-
	(71) Surface (µm 2 )	3750.9 ± 760.4 2142.6 ± 436.5 3094.7 ± 1015 2184.3 ± 335.2 2342.4 ± 459.6
			-	-	-	-	-
	(72) Volume (µm 3 )	550.6 ± 190.7	199.9 ± 47.2	385.1 ± 70.8	180.6 ± 26.4	253.0 ± 53.6
			1>2a	2a<1	-	-	-

Acknowledgments

This work was supported by a grant from ANR-12-JSV4-001-01, the Centre National de la Recherche Scientifique (CNRS), the French Institute of Health and Medical Research (Inserm) and ESPCI ParisTech. We are grateful to Judith Pineau and Aude Duhamel for technical support. The authors declare no competing financial interests.

Diffusion of the neuronal tracer (biocytin) contained in the patch pipette during electrophysiological recordings allowed, after the fixation of slices and diaminobenzidine (DAB)-based processing to visualize the recorded neurons. Dendritic morphologies of the previously physiologically characterized neurons were successfully revealed for 110 out of the 289 recorded neurons. Their morphological variables were extracted from 3D Neurolucida reconstructions (Table 8, Fig. 3A).

Cluster 2a

(n = 26)

Cluster 2b

(n = 11)

Cluster 3a

(n = 20)

Cluster 3b

(n = 29) (50) Nb of dendrites 3.5 ± 0.2 3.7 ± 0.2 4.0 ± 0.5 3.9 ± 0.2 3.6 ± 0.2 (55) Highest order 5.9 ± 0.5 5.6 ± 0.5 5.8 ± 0.5 6.2 ± 0.8 4.8 ± 0.4 segment We examined and compared the dendritic properties of VLPO neurons among the 5 neuronal clusters (Table 8). In particular, we found that neurons from cluster 2b are distinguished from other neurons by their bigger dendritic volume (53). Although not statistically different from all other clusters, neurons from cluster 3b were also found to be smaller (51-53), with in particular a shorter dendritic sholl at 300 µm (60; Table 8).

Axonal properties of VLPO neurons were also extracted from Neurolucida reconstructions when the total axonal length of the reconstructed neuron exceeded 1 mm (Table 9). In some


Si Chen

Andrés Fernando Cardozo Perez

Carine Julcour-Lebigue

Jean-François Blanco

Laurie Barthe

Florence Gayet

Muriel Lansalot

Henri Franck D'agosto

Eric Delmas

Manoury

Andrés F Cardozo

Carine Julcour

Franck D'agosto

Henri Delmas

Eric Manoury
email: eric.manoury@lcc-toulouse.fr

Rinaldo Poli
email: rinaldo.poli@lcc-toulouse.fr

Amphiphilic core-cross

Keywords:

come

Introduction

The power of controlled radical polymerization and the wide choice of monomers and polymerization mechanisms have made it possible to design and synthesize quite complex functionalized macromolecular architectures that were unimaginable only 20 years ago [START_REF] Matyjaszewski | Macromolecular Engineering: Precise Synthesis, Materials Properties, Applications[END_REF]. Among many possible uses of functional polymers, the area of nanoreactors for catalytic applications has developed rather recently but is now rapidly expanding [START_REF] Welsch | [END_REF][3][START_REF] Terashima | Polymer Microgels for Catalysis[END_REF]. By careful design, it is now possible to generate macromolecular architectures where the catalyst is confined in a specific part of the macromolecule conferring specific properties to the catalyst environment (affinity for the reaction substrate, size selectivity, site confinement, etc.) while other parts of the macromolecule are responsible for the nanoreactor compatibility with its environment (homogeneous dispersion, response to stimuli such as heat, pH, magnetic fields etc.). As notable examples, Fréchet et al. have shown the principle of catalyst site confinement for a cascade organic transformation involving one acid-catalyzed and one base-catalyzed step [START_REF] Chi | [END_REF], O'Reilly et al. have turned self-assembled catalytic micelles into unimolecular polymer particles by shell-cross-linking [6], Sawamoto et al. have built thermoresponsive phosphinecontaining microgels and applied them to a few transformations under aqueous biphasic conditions [7,8], and Resmini et al. have used the principle of molecular imprinting to develop shape-selective catalytic nanogels [9,10]. In most cases, the developed nanoreactors were applied to catalytic transformations under homogeneous conditions. Occasionally, the catalyst was recovered and recycled by ultrafiltration, by precipitation, or by thermoregulation.

Efficient catalyst recovery and recycling is of great interest in industrial homogeneous catalysis, particularly when using expensive metals and/or ligand systems, the aqueous biphasic approach being the most attractive one because of the simplicity of the necessary equipment and operating procedures [11]. The prime example of successful industrial application is the Rhone-Poulenc/Ruhrchemie hydroformylation of propene [12], where the Rh catalyst is completely confined in the aqueous phase by triphenylphosphine trisulfonate (TPPTS), but this process is unfortunately inefficient for the higher olefins because of their insufficient water solubility. Among the investigated strategies for circumventing this problem, micellar catalysis is the most attractive one.

Core-functionalized micelles, resulting from the self-assembly of surfactants or amphiphilic diblock copolymers where the catalyst is anchored to the hydrophobic part, are the simplest possible type of nanoreactor and many applications of biphasic catalysis have been described [13][14][15][16][START_REF] Khan | Micellar Catalysis[END_REF][START_REF] Zhang | [END_REF]. The dynamic nature of micelles, however, results in two major obstacles to large scale industrial implementation: uncontrolled swelling leading to the formation of stable emulsions and loss of the free surfactants, even when the critical micelle concentration (CMC) is very low [19][20][21][22][23][24]. For instance, anchoring of a rhodium complex to a poly(norbornene)-based amphiphilic diblock copolymer with CMC = 2.2•10 -6 M and application to the aqueous biphasic 1-octene hydroformylation led to excellent turn over frequency (TOF) but also to leaching with 9 ppm of Rh detected in the organic product phase [25], which is an intolerably high loss for large scale production.

In order to remove both problems, we have recently introduced a new approach, which consists of cross-linking amphiphilic block copolymer micelles at the core to generate unimolecular nano-objects [26,27]. These core-cross-linked micelles (CCM) have been assembled by an efficient one-pot procedure by a polymerization-induced self-assembly (PISA) in water [28,29], using reversible addition-fragmentation chain transfer (RAFT) polymerization in a convergent approach. The ligand needed to bind the catalytic metal was incorporated statistically as a phosphine functionalized comonomer, 4-diphenylphosphinostyrene (DPPS), at low loading (5-25% molar) in the hydrophobic core. This monomer carries a triphenylphosphine (TPP) ligand for metal coordination. The resulting TPP@CCM latex could readily be charged with the hydroformylation precatalyst, [Rh(acac)(CO)2], and the resulting catalytic nanoreactors, [Rh(acac)(CO)(TPP)]@CCM, were successfully used in the aqueous biphasic hydroformylation of 1-octene with excellent activity, catalyst recyclability, and low metal leaching (down to 1.8 ppm) [26].

On the basis of this initial success, we have embarked in a more systematic study of the CCM approach to aqueous biphasic catalysis. Initial questions concerned the modification of the CCM structure in terms of catalyst density and size of the hydrophobic core and the hydrophilic shell (degree of polymerization) and how such changes affect the catalytic performance [27]. Another point of interest is access to CCMs with other ligand functionalities in the hydrophobic core for wider applications in catalysis. In this contribution, we report the synthesis of a CCM functionalized with the bis(4-methoxyphenyl)phenylphosphine (BMOPPP) ligand, BMOPPP@CCM, its physical characterization, its coordination chemistry with [Rh(acac)(CO)2], and the application of the [Rh(acac)(CO)2]-loaded nanoreactors, [Rh(acac)(CO)(BMOPPP)]@CCM, to aqueous biphasic hydroformylation. This specific ligand was chosen for the first CCM chemical modification because of its structural similarity with TPP and consequently of the expected similar polymerization behavior, relative to DPPS, of the suitable ligand-functionalized monomer, 4-[bis(4'-methoxyphenyl)phosphino]styrene (BMOPPS). While the Rh activity in the presence of p-OMe-substituted arylphosphines is very similar to that in the presence of the unsubstituted analogues for hydroformylations conducted with soluble complexes under homogeneous or biphasic conditions [30,31], the p-OMe substitution increases the phosphine binding ability toward Rh [32]. Therefore, lower leaching may be expected if the leaching mechanism involves loss of metal from the nanoreactor core.

Experimental

Materials

All manipulations were performed under an inert atmosphere of dry argon by using Schlenk line techniques. 4,4'-azobis(4-cyanopentanoic acid) (ACPA, >98%, Fluka), methacrylic acid (MAA, 99.5%, Acros), poly(ethylene oxide) methyl ether methacrylate (PEOMA, Mn = 950 g mol -1 , Aldrich), di(ethylene glycol) dimethacrylate (DEGDMA, 95%, Aldrich), 1,3,5-trioxane (Aldrich, > 99%), acetylacetonatodicarbonyl rhodium(I), ([Rh(acac)(CO)2], 99% Strem), chloro(1,5-cyclooctadiene) rhodium(I) dimer ([Rh(COD)Cl]2, 98%, Strem), triphenylphosphine (PPh3 or TPP, >98.5%, Fluka), chlorobis(4-methoxyphenyl) phosphine, (>98%, Alfa), and 4-bromostyrene (98%, stab. with 0.1% 4-tert-butylcatechol, Alfa) were used as received. Styrene (S, 99%, Acros) was purified by passing through a column of active basic aluminium oxide to remove the stabilizer. The RAFT agent 4-cyano-4thiothiopropylsulfanyl pentanoic acid (CTPPA) was synthesized as described previously [33].

Mg turnings was washed with HCl (1M) until the metallic colour appearing then washed with diethyl ether. Solvents were dried by standard procedures and distilled under argon prior to use.

2.2.Characterization techniques 2.2.1. Nuclear Magnetic Resonance

1 H NMR and 31 P NMR spectra were recorded in 5 mm diameter tubes at 297 K in D2O, DMSO-d6 or THF-d8 solution (the polymerization medium aliquots were directly dispersed in the solvent) using a Bruker Avance 400 spectrometer. 1 H and 13 C chemical shifts were determined using the residual peak of deuterated solvent as internal standard and are reported in ppm (δ) relative to tetramethylsilane. 31 P chemical shifts are reported relative to external 85% H3PO4. The solid-state 13 C NMR experiment was recorded on a Bruker Avance 400 spectrometer equipped with a 3.2 mm probe. The sample was spun at 16 kHz at the magic angle using ZrO2 rotors, using a small flip angle (~30°) with a recycle delay of 5 s and a contact time of 2 ms. Peaks are labelled as singlet (s), doublet (d), triplet (t), multiplet (m) and broad (br).

The aromatic C positions are labeled as Ci (ipso, P bonded), Co (ortho), Cm (meta) and Cp (para, bonded to OMe or to CH=CH2). For the CCM characterization, the chemical shift scale was calibrated on the basis of the solvent peak (δ 2.50 for DMSO, 3.58 and 1.73 for THF), and 1,3,5-trioxane was used as an integration reference (δ 5.20).

Size Exclusion Chromatography

Size exclusion chromatography (SEC) measurements were performed in THF (with butylhydroxytoluene (BHT) as a flow rate marker) at 20 °C with a flow rate of 1.0 mL min -1 .

All polymers were analyzed at a concentration around 5 mg mL -1 after filtration through a 0.45 µm pore-size membrane. The separation was carried out on a precolumn and three columns in series (type Styragel HR1 / HR3 / HR4). A multi-angle diffusion light scattering (Mini Dawn TriStar Wyatt) was used as detector coupled with a Wyatt Optilab Rex refractometer.

Dynamic Light Scattering

The intensity-average diameters of the latex particles (Dz) and the dispersity factor (PDI, polydispersity index) were measured at 25 °C on a Malvern Zetasizer NanoZS. After filtration through a 0.45 µm pore-size membrane, deionized water or THF was used to dilute the latex sample. Solutions were analyzed without further filtration to ensure that undesired populations were not removed. Data were analyzed by the general-purpose non-negative least squares (NNLS) method. The typical accuracy for these measurements was 10-15%.

Transmission Electron Microscopy

The morphological analysis of the copolymer nano-objects was performed with a JEOL JEM 1011 transmission electron microscope equipped with 100kV voltage acceleration and tungsten filament (Service Commun de Microscopie Electronique TEMSCAN, plateforme de l'Université Paul Sabatier, Toulouse, France). Diluted latex samples were dropped on a formvar/carbon-coated copper grid and dried under vacuum.

Mass Spectrometry

The mass spectral analyses were performed with a high resolution electrospray XevoG2QT Waters instrument by "Service Commun de Spectrométrie de Masse" of the Université Paul-Sabatier, Toulouse, France.

Synthesis of 4-[bis(4'-methoxyphenyl)phosphino]styrene (BMOPPS)

Chlorobis(4-methoxyphenyl)phosphine (1g, 3.56 mmol) was added slowly at 0 °C to a solution of the Grignard reagent prepared from 4-bromostyrene (0.65 g, 3.56 mmol) and Mg (0.104 g, 4.275 mmol) in dry THF (4 mL). After the addition was complete, the reaction mixture was stirred at room temperature for 3 h. The reaction mixture was then diluted with ethyl acetate (30 mL) and washed sequentially with water (2x50 mL), 10% aqueous HCl (2x50 mL), saturated aqueous NaHCO3 (2x50 mL), and brine (2x50 mL). The organic layer was dried over MgSO4, filtered, and concentrated in vacuum. The crude product was purified by silica gel chromatography (5% Et2O/hexane, then Et2O) to afford 1 as a white solid (0.66 g, 53%). 1 H NMR (400 MHz, CDCl3): δ 7.39-7.22 (m, 8H, CHAr), 6.93-6.90 (m, 4H, CHAr), 6.72 (dd, 1H, J = 10.9 Hz, J = 17.6 Hz, CH), 5.78 (d, 1H, J = 17.6 Hz, CH2), 5.29 (d, 1H, J = 10.9 Hz, CH2), 3.83 (s, 6H, CH3 OMe). 31 P{ 1 H} NMR (162 MHz, CDCl3, 298K): δ -8.93. 13

Preparation of the BMOPPP@CCM latex by one-pot RAFT polymerization in water.

Step 1: Preparation of the P(MAA-co-PEOMA)-TTC macromolecular RAFT agent (macroRAFT) in water.

A stock solution containing ACPA (30 mg mL -1 ) in deionized water (1 mL) containing also NaHCO3 (30 mg) was prepared. 100 µL of this stock solution (3 mg ACPA, 0.0108 mmol), 15 mg of CTPPA (0.054 mmol), 75 mg of MAA (0.87 mmol), 0.79 g of PEOMA (0.83 mmol) and 4.2 g of deionized water (including the water amount of the ACPA solution) were added into a 25 mL flask equipped with a magnetic stirrer bar, which was then sealed with a rubber septum. 1,3,5-trioxane was also added into the flask as an internal reference for the determination of the monomer conversion by 1 H NMR. The solution was purged for 45 min with argon and then heated to 80 °C in a thermostated oil bath with stirring. After 120 min, 0.15 mL of solution was taken to determine the monomer conversion and the molar mass of the macroRAFT product.

The overall monomer molar conversion was about 98% as determined by 1 H NMR spectroscopy in DMSO-d6. The molar mass was analyzed by size exclusion chromatography (SEC) in THF (experimental Mn = 11200 g mol -1 ; Ɖ = 1.19).

Step 2: Chain extension of the macroRAFT with S and BMOPPS in water.

During Step 1, a suspension containing 1.57 g of S (15 mmol), 0.269 g of BMOPPS (0.772 mmol, 5%mol relative to S) in 4.62 g of deionized water, to which was also added the ACPA stock solution (100 µL containing 3 mg of ACPA, 0.0108 mmol) were purged separately for 45 min with an argon stream at 0 °C. This mixture was quickly injected into the first flask under argon at 80 °C after the end of Step 1. The separated organic/aqueous phases became one opaque suspension phase at around 1h. After 2 hours of further stirring for the resulting suspension, a 0.5 mL sample was withdrawn for analysis and the polymerization was quenched by immersion of the flask in iced water. The overall conversion of S (94%) was determined by gravimetric analysis and that of BMOPPS (100%) was measured by 31 P NMR in THF-d8. SEC: Mn = 43900 g mol -1 (Ɖ = 1.3). Dz (DLS) = 78 nm (PDI = 0.2).

Step 3: Cross-linking.

In the flask containing the P(MAA-co-PEOMA)-b-P(S-co-BMOPPS) latex from Step 2, 0.48 g of S (4.6 mmol), 0.137 g of DEGDMA (0.565 mmol, 10%mol relative to S), 90 µL of the ACPA stock solution (2.7 mg of ACPA, 0.009 mmol) and 2.43 g of deionized water were further added. The mixture was purged for 1 h with argon at 0 °C, and the flask was then placed in an oil bath thermostated at 80 °C. After 90 min, the polymerization was quenched by immersion of the flask in iced water. The overall conversion of the comonomers (97%) was determined by 1 H NMR and 31 P NMR in THF-d8. DLS (H2O): Dz = 81 nm (PDI = 0.16). DLS (THF): Dz = 207 nm (PDI = 0.20).

Metal complexation to the phosphine ligand within the nanoparticle core.

[Rh(acac)(CO)2] (31.7 mg, 1.01 eq) in toluene (1 mL) was added to a previously swollen nanoparticles latex (5 mL, 0.1 mg mL -1 ) prepared from the BMOPPP@CCM latex (2 mL) diluted in D2O (3 mL) by addition of toluene (0.5 mL). The swelling was very rapid (< 1 min upon stirring at room temperature) as confirmed by visual disappearance of the toluene phase and by the 31 P NMR observation of the core phosphine resonance (see Results and Discussion).

The reaction mixture was stirred at room temperature for 10 min until the latex color changed to yellow. The resulting latex was washed by toluene (2x1 mL) under argon to remove any excess of the Rh precursor; both toluene washings were colorless. The [Rh(acac)(CO)(BMOPPP)]@CCM latex was collected after decantation for further NMR studies. 31

Biphasic hydroformylation catalysis

The catalytic aqueous phase was prepared under a nitrogen atmosphere by diluting the CCM latex in Milli-Q water (for a phosphine equivalent of 0.65 mmol in 25 mL), then swelling the hydrophobic nanoparticle core with 3 mL of decanal, prior to the addition of the Rh precursor, [Rh(acac)(CO)2] (43 mg, 0.163 mmol), dissolved in 3 mL of decanal. At each step, the resulting mixture was vigorously stirred for a few minutes. This colloidal suspension was poured into the autoclave, then immediately covered by additional decanal (56 mL) and by 1octene (81.6 mmol). The reactor was flushed three times with 15 bar of nitrogen, then four times with 15 bar of syngas. It was subsequently heated under low syngas pressure (2 bar) and slow stirring speed (300 rpm, well below gas self-induction) to generate the catalytic species in situ, albeit hindering the start of the reaction. When the desired reaction temperature (363 K) was achieved (after about half an hour), stirring was stopped and the autoclave was pressurized and constantly fed with syngas at the desired pressure (20 bar). A sample was withdrawn to evaluate the amount of products formed during the heating period.

Then, the data acquisition was started and the stirring speed was set to 1200 rpm. Both temperature and pressure of the reactor and the gas ballast were recorded on-line, in order to measure the instantaneous syngas consumption. After a few hours of reaction, a final sample of the organic phase was withdrawn for the chromatographic analysis (using anisole as internal standard). Acquisition and heating were stopped and the autoclave was cooled slowly at low stirring speed (200 rpm). Once the autoclave was cold, stirring was stopped and the reactor was depressurized and purged four times with nitrogen. The whole reaction mixture was left to settle overnight under a nitrogen atmosphere. Finally, the contents of the autoclave were then taken out and separated. An aliquot of the recovered organic phase was diluted into water (with a volumetric dilution factor of 10 5 ) for the Rh ICP/MS analysis.

Results and Discussion

Monomer and copolymer synthesis

The BMOPPP@CCM latex was prepared by an analogous procedure to that recently described for the similar TPP@CCM latex [26], by replacing the DPPS comonomer with the related 4-[bis(4'-methyoxyphenyl)phosphine]styrene (BMOPPS). This new ligand-functionalized monomer was prepared from chlorobis(4-methoxyphenyl)phosphine and 4-bromostyrene following the same procedure reported for the synthesis of DPPS (see Scheme 1) [34]. The spectroscopic properties are entirely as expected, with the 31 P{ 1 H} NMR resonance (δ -8.93 in CDCl3) only slightly upfield shifted from that of DPPS, the protons of the vinyl group yielding a characteristic pattern in the 6.7-5.3 region of the 1 H-NMR spectrum, and the corresponding C nuclei yielding resonances at δ 136.5 and 114.4 in the 13 C{ 1 H} NMR spectrum, which is shown in full in the SI (Figure S1). Scheme 1. Procedure used for the synthesis of BMOPPS.

The BMOPPP@CCM latex was obtained by a one-pot three-step synthesis, based on the RAFT methodology, as described in Scheme 2. The kinetics of each step is illustrated in Figure 1. In the first step, a 50:50 mixture of methacrylic acid (MAA) and poly(ethylene oxide) methyl ether methacrylate (PEOMA) with an average of 19 ethylene oxide units was statistically copolymerized using 4-cyano-4-thiothiopropylsulfanyl pentanoic acid (CTPPA) as the controlling agent and 4,4'-azobis(4-cyanopentanoic acid) (ACPA) as the radical source. Full conversion was attained within 2 h (see Figure 1, left). The overall monomer/controlling agent ratio being set at 30, the polymer chains obtained at complete conversion have an experimentally determined (SEC analysis) number average molar mass, Mn = 11200 g mol -1 , not far from the expected 30 monomer units per chain (Mn,th = 15625 g mol -1 ) and a low dispersity (Ɖ = 1.19). The resulting water-soluble HOOCCH2CH2C(CN)(CH3)-P(MAA15-co-PEOMA15)-SC(S)SPr was used in step 2 as macro-controlling agent, upon further addition of ACPA, for the chain extension with the mixture of styrene (S) and BMOPPS (95:5 molar ratio corresponding to 300 monomer units per chain). During this step, the solid BMOPPS monomer is dissolved in the styrene yielding a single liquid phase that is added to water. The emulsion polymerization starts rather slowly with an induction period (Figure 1, center) that corresponds to the time required for the P(MAA15-co-PEOMA15) hydrophilic chains to add sufficient hydrophobic monomer units to start to self-assemble. Once micelles are formed, the polymerization is fast and a complete conversion is obtained within 2 h. The polymer SEC analysis confirms the efficiency and control of the chain extension. The experimentally determined molar mass (Mn = 43900 g mol -1 ) of the polymer analyzed after this step increased relative to the starting P(MAA15-co-PEOMA15) chains while the molar mass distribution remained narrow (Ɖ = 1.3) although slightly broader than for P(MAA15-co-PEOMA15) (Ɖ = 1. 19). This appears related to the presence of a certain amount of dead P(MAA15-co-PEOMA15) chains, see Figure S2 in the Supporting Information. Nevertheless, P(MAA-co-POEMA)-b-P(S-co-BMOPPP) amphiphilic block copolymer micelles exhibiting narrowly distributed sizes were obtained after step 2 (see characterization below). Using a proportion of the phosphinefunctionalized monomer greater than 5% results in a three-phase system with undissolved solid BMOPPS and the polymerization does not yield well-dispersed micelles of narrow size distribution. Thus, the chemistry involving BMOPPP is less flexible in terms of degree of ligand functionality relative to the previously reported TPP@CCM synthesis, where the hydrophobic core could be charged with up to 25% of the phosphine-functionalized monomer DPPS [26,27]. The resulting solution was then treated in a third step with a 90:10 molar mixture of styrene and di(ethylene glycol) dimethacrylate (DEGDMA) used as cross-linking agent. This step takes place very rapidly without induction time (Figure 1, right) to yield the desired BMOPPP@CCM. Higher content of DEGDMA would not induce the formation of individual core-shell nano-objects but rather a macrogel formation, as described previously [26].

BMOPPP@CCM characterization

The full 1 H and 31 P{ 1 H} NMR characterization was carried out in THF-d8, which is a good solvent for all the polymer constituents. The 1 H NMR spectrum, see Figure 2a, clearly shows the core aromatic protons in the δ 7.5-6.2 region. The shell PEO side chains give rise to a sharp resonance at δ 3.63 (overlapped with one of the two THF-d8 residual proton resonances)

for the CH2 protons and a smaller resonance at δ 3.34 for the OMe chain end. The backbone aliphatic protons of both core and shell are visible as two broad features at δ 2.2-1.2. The aromatic protons of the BMOPPP moiety overlap with those of PS. The BMOPPP OMe protons probably give rise to the small resonance at δ 3.82, to the left of the large PEO CH2 resonance (cf. δ 3.83 for the monomer in CDCl3), providing direct evidence for the incorporation of BMOPPS in the CCM. Indeed, this peak is absent in the 1 H NMR spectrum of the analogous TPP@CCM, the two spectra being otherwise identical [26]. The extensive resonance overlap hampers the quantitative measurement of the core and shell monomers, however more clear information was obtained from the NMR of the swollen latex (next section). Furthermore, a solid state 13 C{ 1 H} MAS-NMR analysis with quantitative integration reveals the correct area ratio expected for the resonances of the aromatic C atoms at δ 125-130 and of the PEG C atoms at δ 70 (see SI, Figure S3). Further analyses were performed by 31 P{ 1 H} NMR (Figure 2b), which confirmed the incorporation of the phosphine functionalized styrene monomer in the CCM. The phosphorus resonance of the BMOPPS monomer at δ -8.9 is slightly broadened and shifted to δ -11.4 once incorporated in the polymer structure. This displacement was a convenient probe to monitor the BMOPPS consumption during the polymerization. Additional NMR investigations will be shown below, in reference to the complexation studies.

Figure 2. a) 1 H NMR spectrum of BMOPPP@CCM in THF-d8. The resonance marked with ○ belongs to H2O, that marked with □ to silicone grease and those marked with * to the THF-d8 residual proton resonances. b) 31 P{ 1 H} NMR spectra of the BMOPPS monomer (blue) and of BMOPPP@CCM (brown) in THF-d8.

The particle size was measured by DLS in water both before and after cross-linking, as well as in THF for the final cross-linked BMOPPP@CCM particles. The Dz values are ca. 80 nm in water for both the micelles and the CCMs, with a narrow size distribution (PDI ca. 0.2).

The CCMs swelled by a factor of ca. 2.5 in diameter (17 in volume) when placed in a THF solution, see Figure S4 in the SI (cf. 10 in volume for the related TPP@CCM, both with 5% and with 10% molar fraction of phosphine functionalized monomer in the core [26,27]). The spherical morphology, dimensions, and narrow polydispersity were in all cases confirmed by TEM observations (Figure 3). Swelling of the CCM latex by toluene leads to several changes in the 1 H NMR spectrum, see Figure 4(b). A first one is the reappearance of the core constituents as broad features at δ 6-7 for the aromatic protons (Figure 4B) and at δ 1-2 for the backbone protons (Figure 4D). These broader resonances overlap with the sharper ones of the free toluene located inside the CCM core (only the latex phase was carefully selected for the NMR measurement after swelling and decanting), at δ 6.5-6.8 (aromatic) and 1.75 (methyl). The small resonance at δ 2.9 (see Figure 4C) is assigned to the BMOPPP methoxy protons. Although this resonance is upfield shifted by almost 1 ppm from the value in the monomer and in the THF-d8-solvated polymer (vide supra), which may be related to the different solvation, there are no other resonances expected for the polymer in this region and the integrated intensity relative to the PEO peaks is in relatively good agreement with the expected value (observed ratio of 0.061 vs. a theoretical value of 0.076). For comparison, the OMe resonance in the BMOPPS monomer also shifts upfield from δ to 3.27 when the solvent from CDCl3 to toluene-d8. Note that no backbone resonance is visible for the unswollen sample, suggesting that only the PEO chains are solvated by water, whereas the polymer chain backbone and the MAA methyl groups of the shell compartment remain solidary with the hydrophobic core in the pristine (unswollen) latex.

The core phosphine functions become equally visible, after swelling, by 31 core solvation by toluene makes this environment compatible with the PEO chains. The deconvolution of the PEO CH2 resonance as the sum of two Lorentzian functions (see details in the SI, Figure S5) yields a chain distribution of (23.2±0.1)% in water and (76.8±0.1)% in the core. This illustrates a slightly greater tendency of core confinement for toluene-swollen BMOPPP@CCM than for the analogous TPP@CCM (10% of triphenylphosphine molar content in the core), for which the water/core ratio was determined by the same technique as 30.3:69.7(±0.1)) [26].

Integration of the 1 H spectrum for the toluene-swollen sample affords the expected intensities of the aromatic and backbone protons relative to the PEO protons when considering only the protons in the flexible chains (i.e. excluding the cross-linked nucleus, presumably characterized by slow tumbling even after swelling) and also allows a rough estimate of the amount of toluene, although these measurements are quite imprecise because of overlap. The solvent amount is in the range of 730-810 molecules per chain, depending on whether the calculation is based on the Me or the aromatic resonance intensity. This amount is similar to that measured for the swelling of the related TPP@CCM [26].

After swelling, the hydroformylation precatalyst, [Rh(acac)(CO)2], could be readily introduced into the CCM core by stirring the latex with a toluene solution of the metal complex, as demonstrated by 31 [35] and 43.5 (JPRh = 175.6 Hz) [36], bracket the resonance attributed to [Rh(acac)(CO)(BMOPPP@CCM)]. When only half of the phosphine ligands are metal bonded (Rh/P = 0.5), however, no signal is visible in the 31 P{ 1 H} NMR spectrum. This behavior is identical to that observed for the analogous [Rh(acac)(CO)(TPP@CCM)] and is assigned to a rapid exchange between the coordinated and the free phosphine ligands [26]. This observation indicates that the exchange rate for the BMOPP ligand, like that of the TPP ligand, is in the appropriate range to yield coalescence at room temperature.

Figure 6. 31 P{ 1 H} NMR spectra of [Rh(acac)(CO)(BMOPPP@CCM)] with different amounts of rhodium complex. The starred resonance is due to a minor amount of oxidized phosphine impurity.

Hydroformylation catalysis

The BMOPPP@CCM latex was investigated as nanoreactor for the biphasic Rh-catalyzed hydroformylation of 1-octene, using the same operating conditions as for the related TPP@CCM [26,27]: 363 K, 20 bar of syngas pressure and 20% molar as initial concentration of 1-octene in decanal. The volume ratio of organic mixture to aqueous latex suspension was set to 3:1 to keep the catalytic phase dispersed into the organic phase after swelling of the CCM.

Table 1 compares the performance of this new ligand in terms of activity, selectivity and Rh leaching with respect to the reference TPP@CCM. As stated in section 3.1, the BMOPPP@CCM latex could only be prepared with a molar fraction of functionalized monomer in the hydrophobic core of 5% (BMOPPS:S = 1:19), whereas the corresponding TPP@CCM latex could also be prepared with higher molar fractions [26]. The most useful comparison in terms of performances related the two CCM with the same functional monomer content [27].

The BMOPPP@CCM based catalyst yielded a quite similar performance as the TPP homologue, with a marginally greater initial TOF and a marginally smaller l/b ratio. Similar activities and selectivities were also reported in homogeneous catalysis in the presence of p-OMe-substituted and non-substituted arylphosphines [30,31]. However, the BMOPPP@CCM based catalyst unexpectedly resulted in twice more rhodium leaching in the organic phase. This leaching does not appear related to loss of rhodium from the nanoreactors. Indeed, it is known that Rh I binds more strongly to P(C6H4-p-OMe)3 than to P(C6H5)3 by 7.0 kcal/mol [32].

Furthermore, we have previously demonstrated by DLS analysis that the recovered organic phase contains significant amounts of nano-objects and that the affinity of the nanoreactors for the organic phase increases with core swelling and aggregation [27]. b Initial reaction rate (with respect to non-swollen aqueous phase) and corresponding TurnOver Frequency calculated from the syngas consumption during the first 5 min of reaction after the gas absorption phase. c Linear to branched aldehyde ratio determined from the GC/FID analysis of the final sample. d Rh concentration in the organic phase measured by ICP/MS. e Results recalled from ref. [27].

The DLS measurement of the organic phase recovered after run 1 (Table 1) yields the result shown in Figure 7. The measurement confirms that there are indeed particles dispersed in the organic phase and that these belong to two separate distributions. A minor distribution with average Dz = 120 nm corresponds to the expected size for the CCM after swelling with the solvent of the catalytic run (decanal, a poorer solvent than THF for the polymer core). The major distribution has a much larger average particle size (average Dz = 950 nm), clearly corresponding to aggregated polymer particles. The much greater size of this distribution and its greater proportion relative to the non-aggregated distribution, with respect to the particles found in the organic phase after catalysis with [Rh(acac)(CO)(TPP@CCM)] [27], gives additional weight to our recent proposition that bigger aggregates become more lipophilic and therefore leaching correlates with the extent of aggregation. Further proof of the presence of nanoparticles in the organic phase is provided by 31 P NMR (see SI, Figure S6).

. DLS of the recovered organic phase from the catalytic run (entry 1, Table 1). The reason for the formation of bigger aggregates with BMOPPP than with TPP can be attributed to the greater affinity of BMOPPP for binding Rh [32]. Since the experiments are run with a high P:Rh ratio in order to have optimum l/b selectivity and since the catalyst exists as equilibrating [RhH(CO)3(phosphine)] and [RhH(CO)2(phosphine)2] species, a greater proportion of the bis-phosphine species will be generated by the better binding BMOPPP, which leads to the greater formation of lipophilic aggregates by interparticle cross-linking. We have not carried out specific recycling experiments for this particular catalytic nanoreactor, since such studies were previously carried out for the related TPP@CCM, which shows lower leaching.

Conclusions

Replacement of diphenylphosphinostyrene with 4-[bis(p-methoxyphenyl)phosphino]styrene in our optimized synthesis of core-cross-linked micelles (CCM) by emulsion RAFT polymerization [26,27] leads to the successful fabrication of functionalized nanoreactors containing covalently linked bis(p-methoxyphenyl)phenylphosphine (BMOPPP) ligands in the hydrophobic core, although incorporation of this ligand functionalized monomer is limited by its low solubility in styrene. These BMOPPP@CCM nanoreactors show the same behavior as the previously described TPP@CCM in terms of core swelling, coordination of [Rh(acac)(CO)2], intra-particle phosphine ligand exchange, and catalytic activity in aqueous biphasic 1-octene hydroformylation. Catalyst leaching, however, is slightly greater, proving the point that this metal leaching process is not related to partial metal loss from the nanoreactor but rather to partial loss of the entire nanoreactor into the organic product phase. This phenomenon is presumably favored by a greater tendency of this more strongly bonding phosphine to form bigger and more lipophilic particle aggregates through interparticle crosslinking. On the basis of these hypotheses, improvement of this catalytic nanoreactor appears possible by turning to polymer-anchored bidentate ligands and efforts to assemble polymer architectures of this type are currently underway.

  C{ 1 H} NMR (101.5 MHz, CDCl3): δ 160.3 (s, Cp(OMe)), 138.2 (d, JC-P =10.1 Hz, Ci, 1C), 137.5 (s, 1C, CpCHCH2), 136.5 (s, 1C, CH=CH2), 135.3 (d, J = 21.3 Hz, Cm(OMe)), 133.3 (d, JC-P = 19.3 Hz, Cm(CHCH2)), 128.2 (d, JC-P = 8.1 Hz, Ci, 2C), 126.2 (d, J C-P = 6.1 Hz, Co(CHCH2)), 114.4 (s, CH=CH2), 114.2 (d, J = 8.1Hz, Co(OMe)), 55.2 (s, OCH3). HR EI-MS calcd for C22H21O2P 348.1356, found 348.1348. M.p.: 123 °C. Elemental analysis for C22H21O2P•0.1CH2Cl2 : C% 74.38, H% 5.99, found C% 74.57, H% 5.51.

  P{ 1 H} NMR (162 MHz, CDCl3, 298K): δ 44.5 (d, J = 176 Hz) for the polymer linked [Rh(acac)(CO)(BMOPPP)] complexes.

Scheme 2 .

 2 Scheme 2. Synthesis of the BMOPPP@CCM latex.

Figure 1 .

 1 Figure 1. Conversion versus time for the three steps of the BMOPPP@CCM latex synthesis depicted in Scheme 2: step 1 (left), step 2 (center) and step 3 (right).

Figure 3 .

 3 Figure 3. TEM images of micelles (after Step 2 of the synthesis), left, and final core-crosslinked micelles (BMOPPP@CCM), right.

3 . 3 .

 33 Swelling and metal coordinationAs shown in the previous section, all nano-object flexible components (shell and core linear arms) are well solvated in THF-d8. The corresponding 1 H NMR spectrum in D-enriched water (obtained upon directly diluting the latex with D2O, Figure4(a)) only shows the watersolvated hydrophilic shell: PEO CH2 resonance at δ 3.63 and small resonance at δ 3.32 for the terminal OCH3 group (better visible in the expansion of Figure4C). The small and irregular shoulder upfield of the more intense CH2 resonance (at ca. δ 3.55) is probably caused by the PEO CH2 groups located close to the shell/core interface. Water is a non-solvent for the polystyrene-based core. The 31 P{ 1 H} spectrum does not show any visible resonance, Figure5(a).

Figure 4 .Figure 5 .

 45 Figure 4. A: 1 H NMR spectrum of BMOPPP@CCM in D2O before (a) and after (b) swelling with toluene. B-D: Expansions in selected regions. The resonance marked with ○ is due to water, while those marked with ∆ belong to the swelling toluene molecules.

  P{ 1 H} NMR with a resonance at δ -9.7, Figure 5(b). A second change caused by swelling in the 1 H NMR spectrum is the splitting of the PEO CH2 and CH3 resonances into a pair of smaller and sharper resonances on one hand, located at the same positions as in the unswollen latex (cf. (a) and (b) in Figure4C) and therefore assigned to water-solvated PEO chains, and a pair of more intense and broader resonances on the other hand, shifted upfield to δ 3.52 (CH2) and 3.20 (CH3). The latter are assigned to PEO chains that are folded back into the swollen core. Hence, the polystyrene

  P{ 1 H} NMR. The reaction leads to replacement of one CO ligand by the polymer-anchored phosphine with formation of [Rh(acac)(CO)(BMOPPP@CCM)], as indicated by the disappearance of the metal-free BMOPPP@CCM resonance at δ -11.4 and its replacement with a doublet at δ 44.5 (JPRh = 176 Hz) when one equivalent of Rh per P atom is introduced in the CCM, see Figure 6. The analogous molecular complex [Rh(acac)(CO)(BMOPPP)] has not been reported to the best of our knowledge, but the resonances of related complexes [Rh(acac)(CO)(TPP)] and [Rh(acac)(CO)(TMOPP)] [TMOPP = tris(4-methoxyphenyl)phosphine], which are reported respectively at δ 48.6 (JPRh = 179.7 Hz)

1 * 1 . 8 a

 118 Table Results of the biphasic hydroformylation of 1-octene.Operating conditions: [Rh(acac)(CO)2] = 6.5•10 -3 kmol•m -3 φaq, [1-octene] = 1.1 kmol•m -3 φorg, P/Rh = 4, Vφorg = 75 mL (octene + decanal), Vφaq = 25 mL (latex + water), T = 363 K, Psyngas = 20 bar (CO/H2 = 1),  = 1200 rpm.

Acknowledgements

We are grateful to the Agence Nationale de la Recherche (ANR) for support of this work through grant "BIPHASNANOCAT" (ANR-11-BS07-025-01). Additional support from the Centre National de la Recherche Scientifique (CNRS) and from the Institut Universitaire de France (IUF) is also gratefully acknowledged.