Cécile Charrier

Patricia Machado

Ry Y Tweedie-Cullen

Isabelle M Mansuy

Antoine Triller
email: triller@biologie.ens.fr

A crosstalk between β1 and β3 integrins controls glycine receptor and gephyrin trafficking at inhibitory synapses

The regulation of glycine receptor (GlyR) number at synapses is fundamental for the efficacy of inhibition and the control of neuronal excitability in the spinal cord. GlyR accumulation at synapses depends on the scaffolding molecule gephyrin and is linked to GlyR synaptic dwell time. However, the mechanisms that tune GlyR synaptic exchanges depending on the neuronal environment are unknown. Integrins are cell adhesion molecules and signaling receptors. Using single quantum dot and FRAP imaging, we demonstrate in rats that β1 and β3 integrins regulate the synaptic dwell time of both GlyRs and gephyrin to adjust synaptic strength. β1 and β3 integrins crosstalk via calcium/calmodulin-dependent protein kinase II and adapt GlyR lateral diffusion and gephyrin-dependent trapping at synapses. This provides a mechanism to maintain or adjust the steady state of postsynaptic molecule exchanges and the level of glycinergic inhibition in response to neuron-and gliaderived signals or extracellular matrix remodeling.

Introduction

While synapses are relatively stable structures, their molecular components are exchanged within short time scales [START_REF] Triller | New concepts in synaptic biology derived from singlemolecule imaging[END_REF] . Postsynaptic scaffolding molecules are renewed in tens of minutes both in vitro and in vivo (e.g. [START_REF] Calamai | Gephyrin oligomerization controls GlyR mobility and synaptic clustering[END_REF][START_REF] Gray | Rapid redistribution of synaptic PSD-95 in the neocortex in vivo[END_REF][START_REF] Specht | The dynamics of synaptic scaffolds[END_REF] ). In the neuronal membrane, recent advances in single particle imaging techniques have enabled the visualization of neurotransmitter receptor lateral diffusion into and out of synapses [START_REF] Triller | New concepts in synaptic biology derived from singlemolecule imaging[END_REF][START_REF] Dahan | Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking[END_REF] .

As receptors are mostly inserted in and removed from the plasma membrane at nonsynaptic sites [START_REF] Triller | New concepts in synaptic biology derived from singlemolecule imaging[END_REF] , lateral diffusion and trapping at synapses are fundamental mechanisms for the sorting and accumulation of receptors at excitatory or inhibitory synapses. Synaptic strength is closely related to the dwell time of the postsynaptic molecules, and both can be adjusted during synapse maturation and plasticity [START_REF] Triller | New concepts in synaptic biology derived from singlemolecule imaging[END_REF][START_REF] Gray | Rapid redistribution of synaptic PSD-95 in the neocortex in vivo[END_REF] .

However, how changes in the neuronal environment are translated into changes in the dwell times of synaptic molecules remains unknown.

Integrins mediate adhesion between the extracellular matrix (ECM) and the cytoskeleton and transduce bidirectional signaling cascades [START_REF] Hynes | Integrins: bidirectional, allosteric signaling machines[END_REF][START_REF] Miranti | Sensing the environment: a historical perspective on integrin signal transduction[END_REF] . They are transmembrane heterodimers composed of one α and one β subunit, which are receptors for ECM molecules, soluble factors and counter-receptors. At least ten integrin subunits are expressed in the central nervous system [START_REF] Pinkstaff | Integrin subunit gene expression is regionally differentiated in adult brain[END_REF] and some are localized at synapses (e.g. [START_REF] Pinkstaff | Integrin subunit gene expression is regionally differentiated in adult brain[END_REF][START_REF] Chan | Integrin requirement for hippocampal synaptic plasticity and spatial memory[END_REF][START_REF] Chavis | Integrins mediate functional pre-and postsynaptic maturation at a hippocampal synapse[END_REF][START_REF] Cingolani | Activity-dependent regulation of synaptic AMPA receptor composition and abundance by beta3 integrins[END_REF] ). Integrins are central elements in neuron-neuron and neuron-glia interactions during synapse maturation, synapse plasticity and after injury [START_REF] Chavis | Integrins mediate functional pre-and postsynaptic maturation at a hippocampal synapse[END_REF][START_REF] Kloss | Integrin family of cell adhesion molecules in the injured brain: regulation and cellular localization in the normal and regenerating mouse facial motor nucleus[END_REF][START_REF] Staubli | An inhibitor of integrin receptors blocks long-term potentiation[END_REF] . Recent studies have highlighted the involvement of the β1 and β3 integrins at excitatory synapses. In hippocampal neurons, β1 integrins are required for long-term potentiation and spatial memory [START_REF] Chan | Integrin requirement for hippocampal synaptic plasticity and spatial memory[END_REF][START_REF] Chan | Beta 1-integrins are required for hippocampal AMPA receptordependent synaptic transmission, synaptic plasticity, and working memory[END_REF][START_REF] Huang | Distinct roles of the beta 1-class integrins at the developing and the mature hippocampal excitatory synapse[END_REF][START_REF] Kramar | Integrin-driven actin polymerization consolidates long-term potentiation[END_REF] . β3 integrins coordinate the maturation of the pre-and post-synaptic compartments and mediate activitydependent regulation of excitatory synaptic strength [START_REF] Chavis | Integrins mediate functional pre-and postsynaptic maturation at a hippocampal synapse[END_REF][START_REF] Cingolani | Activity-dependent regulation of synaptic AMPA receptor composition and abundance by beta3 integrins[END_REF] . The function of integrins at inhibitory synapses remains to be determined.

In this study, we have investigated the function of β1 and β3 integrins at glycinergic synapses in spinal cord neurons. Glycine receptors (GlyRs) play a major role in controlling motor neuron excitability and in processing sensory signals [START_REF] Betz | Glycine receptors: recent insights into their structural organization and functional diversity[END_REF] . We have focused on GlyRs containing the α1 subunit, which predominate in the adult spinal cord [START_REF] Betz | Glycine receptors: recent insights into their structural organization and functional diversity[END_REF] . These receptors are α1/β hetero-pentamers and are stabilized at synapses by direct interaction between the β subunit and gephyrin, the main inhibitory scaffolding protein [START_REF] Specht | The dynamics of synaptic scaffolds[END_REF] . We find that β1 and β3 integrins adjust glycinergic synaptic strength with opposing actions: while β1 integrins increase GlyR numbers at synapses, β3 integrins reduce GlyR numbers and glycinergic synaptic strength.

Using single molecule and bulk approaches, we demonstrate that these effects result from a regulation of both gephyrin exchange and GlyR dwell time at synapses. The opposing actions of β1 and β3 integrins rely on calcium/calmodulin-dependent protein kinase II (CaMKII), which regulates GlyR trapping and accumulation at synapses. We also find that β1 and β3 integrins can be further activated by extracellular factors such as thrombospondin I (TSP1) and fibrinogen that are released after injury. Thus, the crosstalk between β1 and β3 integrins provides a mechanism for the regulation of glycinergic synaptic strength integrating signals from neurons and glia.

Results

Opposite actions of β1 and β3 integrins on synaptic GlyRs

To test the possible involvement of integrins at glycinergic synapses, we blocked integrin function in cultured spinal cord neurons using echistatin, a peptide from viper venom containing an arginine-glycine-aspartate (RGD) motif, which specifically inhibits integrins containing the β1 and β3 subunits [START_REF] Pfaff | Comparison of disintegrins with limited variation in the RGD loop in their binding to purified integrins alpha IIb beta 3, alpha V beta 3 and alpha 5 beta 1 and in cell adhesion inhibition[END_REF] . We analyzed the effect of 1-hr treatment with echistatin (100 nM) on GlyR synaptic clusters by immunocytochemistry, using synapsin I as a presynaptic marker. After echistatin treatment, GlyR clusters were brighter than in control conditions (Fig. 1a-b). GlyR immunoreactivity (IR) at synapses increased to 177 ± 11% of the control (n Ctr = 52, n Echis = 51, p < 0.001, Fig. 1c). Similar results were obtained with a synthetic RGD peptide (n GRGESP = 54, n GRGDSP = 54, p < 0.001, Fig. 1c). Together, this indicates that integrins control GlyR accumulation at synapses.

To discriminate between the effects of β1 and β3 integrins on GlyR clusters, we used monoclonal antibodies that specifically block β1 or β3 integrins. One-hour treatment with β3-blocking antibodies (aβ3, 25 μg/ml) increased GlyR cluster-IR to 198 ± 11% of the control (n Ctr = 60, n aβ3 = 60, p < 0.001, Fig. 2a-b). By contrast, treatment with β1-blocking antibodies (aβ1, 25 μg/ml) reduced GlyR cluster-IR to 57 ± 2% of the control (n Ctr = 60, n aβ1 = 60, p < 0.001). The difference between the effect of RGD peptides and that of aβ1 can be explained by the fact that, depending on the α subunit, some αβ1 heterodimers are not receptors for RGD containing molecules [START_REF] Hynes | Integrins: bidirectional, allosteric signaling machines[END_REF] and are therefore not inhibited by these peptides. One-hour treatment with aβ1 or aβ3 had no effect on synapse density or on synapse size (Supplementary Fig. 1). In line with this, the robust changes in GlyR cluster-IR after aβ1 and aβ3 treatments were not associated with similar modifications of GlyR cluster size (Supplementary Fig. 1). This is consistent with previous reports on synapse number or morphology [START_REF] Cingolani | Activity-dependent regulation of synaptic AMPA receptor composition and abundance by beta3 integrins[END_REF][START_REF] Chan | Beta 1-integrins are required for hippocampal AMPA receptordependent synaptic transmission, synaptic plasticity, and working memory[END_REF][START_REF] Huang | Distinct roles of the beta 1-class integrins at the developing and the mature hippocampal excitatory synapse[END_REF][START_REF] Wang | Extracellular proteolysis by matrix metalloproteinase-9 drives dendritic spine enlargement and long-term potentiation coordinately[END_REF] .

To further substantiate the opposite effects of the β1 and β3 integrin blocking antibodies, we overexpressed truncated forms of β1 and β3 integrins, in which the extracellular domain was replaced by EGFP (CTβ1 and CTβ3, respectively [START_REF] Cingolani | Activity-dependent regulation of synaptic AMPA receptor composition and abundance by beta3 integrins[END_REF] ). The low transfection efficiency allowed us to analyze the effects of postsynaptic integrin blockade, by examining transfected postsynaptic neurons that were not surrounded by transfected axons. After 24 hrs, overexpression of CTβ3 resulted in a robust increase in receptor cluster-associated fluorescence (206 ± 23% of the control) whereas overexpression of CTβ1 had the opposite effect (55 ± 4 % of the control) (n EGFP = 37; n CTb1 = 40; n CTb3 = 38, p < 0.001, Supplementary Fig. 2). The direction and the amplitude of these changes were similar to those observed after 1-hr treatment with the blocking antibodies. Together, these results demonstrate that β1 and β3 integrins regulate GlyR numbers at synapses in opposing directions.

The functional consequences of the regulation of GlyR numbers at synapses were tested with whole-cell patch-clamp recordings. We compared the miniature glycinergic postsynaptic currents (mIPSCs) on the same cells before and after antibody application. This showed that mIPSC amplitudes were increased after β3 integrin inhibition, but reduced after β1 integrin inhibition (Fig. 2c-d), consistent with the effects observed by immunocytochemistry. Twenty minutes after aβ3 application, mIPSC amplitudes were increased to 132 ± 4% of the amplitudes before treatment, whereas they were decreased to 76 ± 4% after aβ1 application (n = 5 for each condition, p < 0.001, Fig. 2e). The coefficient of variation of the mIPSC amplitudes was not modified after aβ1 or aβ3 treatment (Supplementary Fig. 3), indicating that β1 and β3 integrins uniformly scale the synaptic strength of all glycinergic synapses. Moreover, the kinetics of the mIPSCs remained unchanged (Supplementary Fig. 3).

The frequency of the mIPSCs varied between cells from 1.0 ± 0.4 Hz to 7.5 ± 0.3 Hz, but was not modified by aβ1 and aβ3 treatments (statistical test: non significant (NS); Fig. 2f). The effects of aβ3 and aβ1 treatments were not observed with denatured antibodies (data not shown). These data indicate that β1 and β3 integrins scale inhibitory synaptic strength in opposite directions by modifying GlyR number at synapses.

Integrins control GlyR lateral diffusion

We have previously shown that GlyR diffusion-trapping is a major mechanism for the regulation of GlyR number at synapses [START_REF] Dahan | Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking[END_REF][START_REF] Charrier | Cytoskeleton regulation of glycine receptor number at synapses and diffusion in the plasma membrane[END_REF][START_REF] Levi | Homeostatic regulation of synaptic GlyR numbers driven by lateral diffusion[END_REF] . To test the effects of integrin inhibition on GlyR diffusion properties in the neuronal membrane, we stained active presynaptic boutons with FM4-64 and monitored the surface mobility of endogenous GlyRs labeled with quantum dots (GlyR-QDs) in single particle tracking (SPT) experiments. The SPT method provides a high spatial resolution (≈ 10 nm) [START_REF] Dahan | Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking[END_REF] .

Consistent with previous observations [START_REF] Dahan | Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking[END_REF][START_REF] Charrier | Cytoskeleton regulation of glycine receptor number at synapses and diffusion in the plasma membrane[END_REF] , some GlyR-QDs remained at synapses or diffused in the extrasynaptic membrane, while others entered or escaped the synaptic area during the recording session (Fig. 3a). After aβ3 treatment, GlyR-QDs generally explored larger surfaces of the extrasynaptic membrane, but smaller surfaces at synapses. After aβ1 treatment, GlyR-QDs were very mobile within and outside synapses and frequently exchanged between the synaptic and extrasynaptic compartments.

Quantification of these experiments indicated that, in the extrasynaptic membrane, GlyR-QD diffusion coefficients were about twice higher than control values after both aβ3 and aβ1 treatments (n Ctr = 1112, n aβ3 = 626, n aβ1 = 682, p < 0.001, Fig. 3b,d). At synapses however, β3 and β1 integrin blockade had opposite effects: GlyR-QD diffusion coefficients were decreased after aβ3 treatment (n Ctr = 288, n aβ3 = 152, p < 0.01), whereas they were increased after aβ1 treatment (n aβ1 = 176; p < 0.001, Fig. 3c). The diffusion coefficients were decreased to 75% of the control after β3 integrin inhibition and increased to 170% of the control after β1 integrin inhibition (Fig. 3d). Therefore, the effects of β1 and β3 integrin inhibition are comparable at the extrasynaptic membrane but opposite at synapses, indicating distinct regulations in the two compartments.

Integrins control GlyR trapping and dwell time at synapses

Given the effects on GlyR diffusion at synapses, we hypothesized that integrins regulate the level of confinement of synaptic receptors. Therefore, we analyzed the trajectories of GlyR-QDs in more detail (Fig. 4a). As shown by plots of the mean square displacement (MSD), GlyRs were more confined after β3 integrin inhibition, but less confined after β1 integrin inhibition (Fig. 4b). In control conditions, the average diameter of the confinement domain at synapses was 175 ± 9 nm (n = 228 trajectories), consistent with previous data [START_REF] Charrier | Cytoskeleton regulation of glycine receptor number at synapses and diffusion in the plasma membrane[END_REF] . The size of the confinement domain was reduced to 71 ± 5% of the control after β3 integrin inhibition whereas it was enlarged to 150 ± 10 % of the control after β1 integrin inhibition (n aβ3 = 115, n aβ1 = 124, p < 0.001, Fig. 4c). This indicates that β1 integrin inhibition induces a relaxation of the constraints that stabilize the GlyRs at synapses, and therefore facilitates GlyR exit from synapses, whereas β3 integrin inhibition has the opposing action.

Next, we tested whether the changes in GlyR confinement affected the time spent by GlyRs at synapses when they diffuse in the plasma membrane. Receptor dwell time in a synapse may vary between seconds and tens of minutes [START_REF] Dahan | Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking[END_REF] , thus exceeding the duration of our recordings (38.4 s). Therefore, we used a dwell time index to estimate the mean fraction of time spent in a synapse (see Online Methods). The cumulative distribution of this index highlighted two populations of receptors: a 'stable' population corresponding to receptors always detected at synapses during the recording session (dwell time index = 1), and a 'mobile' population corresponding to receptors that exchange between the synaptic and extrasynaptic compartments (dwell time index < 1) (Fig. 4d). β1 integrin inhibition reduced GlyR-QD dwell time index from 0.51 ± 0.02 in control condition to 0.34 ± 0.03, whereas β3 integrin inhibition increased it to 0.60 ± 0.03 (n Ctr = 377, n aβ3 = 205, n aβ1 = 253; aβ3: p < 0.05, aβ1: p < 0.001; Fig. 4e). Thus, GlyRs spent more time at synapses after β3 integrin inhibition and more time in the extrasynaptic membrane after β1 integrin inhibition. Furthermore, we found that integrin-mediated regulation of GlyRs did not depend on GlyR endocytosis, exocytosis, protein synthesis or protein degradation (Supplementary Fig. 4), which contrasts with what has been observed for GluR2-containing AMPA receptors 11 (Supplementary Fig. 4). Together, these data demonstrate that β1 and β3 integrins set the equilibrium between the pools of synaptic and extrasynaptic GlyRs and thereby GlyR number at synapses.

Integrins regulate gephyrin stabilization at synapses

At synapses, GlyRs are transiently stabilized by the gephyrin-based postsynaptic scaffold [START_REF] Specht | The dynamics of synaptic scaffolds[END_REF] . Therefore, we tested whether changes in gephyrin assembly were associated with the regulation of GlyR trapping at synapses. In immunocytochemistry, gephyrin cluster-IR increased to 161 ± 5% of the control value after aβ3 treatment (n Ctr = 60, n aβ3 = 60, p < 0.001), whereas it decreased to 59 ± 3% after aβ1 treatment (n aβ1 = 60, p < 0.001, Fig. 5a-b), indicating that integrins control the level of gephyrin at synapses. These effects were not associated with modification of gephyrin expression and did not depend on protein synthesis or protein degradation (Supplementary Fig. 5), indicating that integrins control the exchange between synaptic and non-synaptic gephyrin molecules.

To examine the effects of β1 and β3 integrin blockade on the synaptic dynamics of gephyrin, we transfected neurons with a gephyrin::Venus construct (VeGe [START_REF] Hanus | Activity-dependent movements of postsynaptic scaffolds at inhibitory synapses[END_REF] ) and performed fluorescence recovery after photobleaching (FRAP) of individual synaptic VeGe clusters (Fig. 5c-d). Over a 20 minute time scale, the recovery curves exhibited two main phases: a 'fast' component corresponding to a population of molecules exchanged in a few seconds with non-synaptic non-bleached molecules, and a 'slow' component corresponding to a population of molecules that could reside in a cluster for tens of minutes (Fig. 5e). Integrin inhibition had a noticeable effect on the fast phase of the recovery, which was more rapid and reached a higher plateau after β1 integrin inhibition but was reduced after β3 integrin inhibition. In contrast, the slow phase was not dramatically altered. Ten minutes after photobleaching, the fluorescence recovery was 50 ± 2% of the initial value in control condition (n = 26, Fig. 5f), which is similar to the recovery obtained with endogenous mRFP-gephyrin clusters from a knock-in mouse [START_REF] Calamai | Gephyrin oligomerization controls GlyR mobility and synaptic clustering[END_REF] . It was increased to 58 ± 2% after β1 integrin inhibition (n = 21, p < 0.01) and reduced to 33 ± 1% after β3 integrin inhibition (n =21, p < 0.001). Therefore, integrins determine the steady state of gephyrin synaptic exchange. Altogether, our data indicate that the modulation of gephyrin synaptic turnover by β1 and β3 integrins controls the dwell time and the number of GlyRs at synapses.

Integrins control GlyR synaptic trapping via CaMKII

In contrast to other adhesion molecules such as cadherins and neuroligins, there is no known direct interaction between integrins and postsynaptic proteins.

Nonetheless, integrins interact with a number of cytoskeleton-related molecules and signaling proteins 7 that might regulate the interactions in the postsynaptic scaffold.

We have previously shown that F-actin disruption increases GlyR lateral diffusion, and reduces the amount of both GlyRs and gephyrin at synapses [START_REF] Charrier | Cytoskeleton regulation of glycine receptor number at synapses and diffusion in the plasma membrane[END_REF] . We therefore investigated the involvement of the actin cytoskeleton in integrin-mediated regulations of GlyR lateral diffusion. We found that F-actin disruption with latrunculin (Lat, 3 μM, 25 min) abolished the effect of β1 and β3 integrin inhibition in the extrasynaptic membrane (n Lat = 484, n Lat+aβ3 = 621, n Lat+aβ1 = 386, statistical tests: NS, Fig. 6a). At synapses, however, latrunculin did not prevent the aβ1-induced increase or the aβ3-induced decrease in GlyR diffusion coefficients (n Lat = 103, n Lat+aβ3 = 185, n Lat+aβ1 = 73, p < 0.001 and p < 0.05, respectively). The effects on GlyR clusters were also maintained after latrunculin treatment: β3 integrin blockade increased GlyR cluster-IR to 167 ± 9%, whereas β1 integrin blockade reduced it to 77 ± 4% (n Lat = 40, n Lat+aβ3 = 40, n Lat+aβ1 = 40; p < 0.001 and p < 0.01, respectively, Fig. 6b). This indicates that the lateral mobility of GlyRs depends on actin in the extrasynaptic membrane, whereas integrins regulate GlyR trapping at synapses via a distinct pathway.

We then examined several kinases involved in integrin signaling. Inhibition of Src kinases did not block the effect of aβ1 or aβ3 on GlyR cluster-IR (Supplementary Fig. 6), suggesting that Src kinases do not contribute to these regulations. PKC inhibition with GF109203X (GFX, 50 nM, 25 min) alone did not significantly modify GlyR lateral diffusion or GlyR accumulation at synapses (Supplementary Fig. 7). However, PKC inhibition prevented the effects of integrin

TSP1 and fibrinogen have opposite effects at synapses

Little is known about the ligands of integrins involved in the regulation of synapse function. We examined the effects of TSP1 and fibrinogen, two known β1 and β3 integrin ligands released after injury [START_REF] Defreitas | Identification of integrin alpha 3 beta 1 as a neuronal thrombospondin receptor mediating neurite outgrowth[END_REF][START_REF] Moller | Regulation of thrombospondin in the regenerating mouse facial motor nucleus[END_REF][START_REF] Lin | Differential regulation of thrombospondin-1 and thrombospondin-2 after focal cerebral ischemia/reperfusion[END_REF][START_REF] Schachtrup | Fibrinogen inhibits neurite outgrowth via beta 3 integrinmediated phosphorylation of the EGF receptor[END_REF] . In our experiments, the β1 integrin ligand TSP1 (2 μg/ml, 1 hr) slowed down GlyR lateral diffusion in the extrasynaptic membrane (n Ctr = 487, n TSP1 = 355, p < 0.001) and stabilized GlyRs at synapses (n Ctr = 159, n TSP1 = 130, p < 0.001, Fig. 7a-b). The changes in diffusion properties were associated with an increased accumulation of GlyRs (Ctr: 100 ± 4%, n = 60; TSP1: 147 ± 5%, n = 60; p < 0.001) and gephyrin at synapses (Ctr: 100 ± 4%, n = 60; TSP1: 150 ± 6%, n = 60; p < 0.001, Fig. 7c). The effects of TSP1 were opposite to those observed after inhibition of β1 integrins and were abolished by prior incubation with aβ1 (Supplementary Fig. 8). This shows that TSP1 increases GlyR trapping through β1 integrins.

The β3 integrin ligand fibrinogen (Fib, 1.5 mg/ml, 1 hr) slowed GlyR lateral mobility in the extrasynaptic membrane and destabilized GlyRs at synapses (extrasynaptic: n Vehicle = 614, n Fib = 413, p < 0.001; synaptic: n Vehicle = 218, n Fib = 152, p < 0.001; Fig. 7d-e). Consistent with this, fibrinogen decreased GlyR and gephyrin synaptic levels to 67 ± 2% and 73 ± 2% of the control values, respectively (GlyR and gephyrin: n Vehicle = 60, n Fib = 60, p < 0.001, Fig. 7f). The effects of fibrinogen were opposite to those observed after inhibition of β3 integrins and were abolished by prior incubation with aβ3 (Supplementary Fig. 8), showing that fibrinogen decreases GlyR trapping via β3 integrins. Taken together, these results strongly suggest that GlyR diffusion-trapping and numbers at synapses can be regulated by extracellular factors released after injury.

Discussion

In this study, we have shown that integrins control glycinergic synaptic strength by regulating the number of GlyRs at synapses. Using SPT with blocking antibodies and known integrin ligands, we demonstrate that β1 integrins decrease GlyR diffusion coefficients and increase GlyR confinement at synapses, whereas β3 integrins have opposing actions. β1 and β3 integrins adjust GlyR dwell time at synapses and shift the equilibrium between the pools of synaptic and extrasynaptic receptors toward an increased and decreased synaptic localization, respectively. Importantly, blocking the effects of integrins on GlyR diffusive properties at synapses abolished the regulation of GlyR postsynaptic accumulation. Furthermore, the effects of integrins were not associated with modification of GlyR surface expression and did not depend on endocytosis, protein synthesis or protein degradation. Together, these data reveal that ®1 and ®3 integrins control GlyR synaptic trapping and residence time, which determine the number of GlyRs at synapses in spinal cord neurons.

It is now well accepted that GlyRs may be associated with gephyrin along the secretion pathway [START_REF] Hanus | Intracellular association of glycine receptor with gephyrin increases its plasma membrane accumulation rate[END_REF] and that a large proportion of GlyRs diffuse into and out of synapses in association with gephyrin [START_REF] Calamai | Gephyrin oligomerization controls GlyR mobility and synaptic clustering[END_REF][START_REF] Ehrensperger | Multiple association states between glycine receptors and gephyrin identified by SPT analysis[END_REF] . This implies that GlyRs can be trapped at synapses by GlyR/gephyrin or gephyrin-gephyrin interactions. Consequently, modulating gephyrin oligomerization is expected to affect the stabilization of both GlyRs and gephyrin molecules at synapses. In line with this, it has been shown that overexpression of gephyrin variants with altered oligomerization properties affects GlyR lateral diffusion and postsynaptic accumulation [START_REF] Calamai | Gephyrin oligomerization controls GlyR mobility and synaptic clustering[END_REF][START_REF] Bedet | Regulation of gephyrin assembly and glycine receptor synaptic stability[END_REF] . Alternatively, the modulation of GlyR/gephyrin interaction may affect GlyR synaptic trapping. It has been shown that gephyrin interaction with its partner Pin1 regulates GlyR/gephyrin binding [START_REF] Zita | Post-phosphorylation prolyl isomerisation of gephyrin represents a mechanism to modulate glycine receptors function[END_REF] . Modifications in the gephyrin-binding sequence of the GlyR β subunit have also been proposed to affect GlyR synaptic localization during activity-dependent homeostatic regulation of glycinergic synapses [START_REF] Levi | Homeostatic regulation of synaptic GlyR numbers driven by lateral diffusion[END_REF] . So far, the modulation of GlyR/gephyrin interaction has not been associated with changes in gephyrin postsynaptic accumulation [START_REF] Levi | Homeostatic regulation of synaptic GlyR numbers driven by lateral diffusion[END_REF][START_REF] Zita | Post-phosphorylation prolyl isomerisation of gephyrin represents a mechanism to modulate glycine receptors function[END_REF] . Our immunocytochemistry and FRAP experiments showed that β1 and β3 integrin inhibition modified both the amount of synaptic gephyrin and the exchange between synaptic and non-synaptic gephyrin molecules. This indicates that β1 and β3 integrins modulate gephyrin oligomerization properties.

The regulation of gephyrin oligomerization alone can account for the effects on GlyR and gephyrin dynamics because GlyRs and gephyrin may be co-trafficked and because gephyrin provides the binding sites for GlyRs at synapses. We conclude that gephyrin exchange at synapses is tightly linked to GlyR stabilization. This provides important insight into the dynamics of synaptic multimolecular assemblies in the control of synaptic strength.

Strikingly, β1 and β3 integrins affected inhibitory synaptic strength in opposite directions. β1 and β3 integrins have been shown to regulate other cell functions differentially. For example, L-type calcium channels are up-regulated by β1 integrins and down-regulated by β3 integrins in arteriolar smooth muscles for vasomodulation [START_REF] Wu | Modulation of calcium current in arteriolar smooth muscle by alphav beta3 and alpha5 beta1 integrin ligands[END_REF] . In macrophages, β1-mediated phagocytosis and migration (but not adhesion) are inhibited by β3 integrins [START_REF] Blystone | A molecular mechanism of integrin crosstalk: alphavbeta3 suppression of calcium/calmodulin-dependent protein kinase II regulates alpha5beta1 function[END_REF] . This suggests that the functional counteraction between β1 and β3 integrins may be a general mechanism to adapt cellular responses to the extracellular environment. We performed experiments aimed at identifying the molecular basis of the opposing actions of β1 and β3 integrins at glycinergic synapses. We found that PKC was necessary for integrin effects in and outside synapses, consistent with the requirement of PKC for integrin activation [START_REF] Miranti | Sensing the environment: a historical perspective on integrin signal transduction[END_REF] . We also found that CaMKII was responsible for β1 and β3 integrin actions specifically at synapses. CaMKII inhibition abolished the effects of integrin blockade at synapses but not in the extrasynaptic membrane. Furthermore, CaMKII inhibition increased GlyR mobility at synapses and strongly decreased GlyR synaptic localization. These effects were similar to those observed after β1 integrin inhibition and after β3 integrin activation with fibrinogen. Consistent with this, β1 and β3 integrins have been shown to activate and inhibit CaMKII, respectively [START_REF] Blystone | A molecular mechanism of integrin crosstalk: alphavbeta3 suppression of calcium/calmodulin-dependent protein kinase II regulates alpha5beta1 function[END_REF] . Together, these data indicate that β1 and β3 integrins crosstalk via CaMKII to adjust GlyR trapping at synapses depending on the neuronal environment (Supplementary Fig.

9).

Our results reveal that CaMKII stabilizes GlyRs at inhibitory synapses. By contrast, CaMKII has been shown to destabilize PSD-95 at excitatory synapses [START_REF] Steiner | Destabilization of the postsynaptic density by PSD-95 serine 73 phosphorylation inhibits spine growth and synaptic plasticity[END_REF] and to inhibit high order complex formation between Homer and Shank in vitro [START_REF] Hayashi | The postsynaptic density proteins Homer and Shank form a polymeric network structure[END_REF] . This suggests that CaMKII regulates the dynamics of excitatory and inhibitory postsynaptic membranes in opposite directions to control neuronal activity. An important issue will be to identify the molecular targets of CaMKII for the regulation of inhibitory postsynaptic assembly dynamics. One possibility is that CaMKII directly targets gephyrin. Indeed, using in vitro phosphorylation tests and mass spectrometry, we found that CaMKII can directly phosphorylate gephyrin on at least six serine residues (Supplementary Fig. 10). Three of these sites have already been found to be phosphorylated in vivo (S296 [START_REF] Trinidad | Comprehensive identification of phosphorylation sites in postsynaptic density preparations[END_REF] , S318 [START_REF] Trinidad | Comprehensive identification of phosphorylation sites in postsynaptic density preparations[END_REF] , S337 [START_REF] Tweedie-Cullen | Comprehensive mapping of posttranslational modifications on synaptic, nuclear, and histone proteins in the adult mouse brain[END_REF] ), suggesting that they are of physiological relevance. The sites we identified here are mainly located at the junction between the linker region and the C-terminal domain of gephyrin, which dimerizes and is essential for the formation of higher order oligomers (see [START_REF] Bedet | Regulation of gephyrin assembly and glycine receptor synaptic stability[END_REF][START_REF] Saiyed | Molecular basis of gephyrin clustering at inhibitory synapses: role of G-and E-domain interactions[END_REF] , Supplementary Fig. 10). Further studies are required to determine whether β1 and β3 integrins modulate the phosphorylation state of these sites in living spinal cord neurons and how the multiple phosphorylation of gephyrin modulates its oligomerization properties.

Our results showed that β1 and β3 integrins modulate inhibitory synapses in response to TSP1 or fibrinogen, two molecules released after injury [START_REF] Moller | Regulation of thrombospondin in the regenerating mouse facial motor nucleus[END_REF][START_REF] Lin | Differential regulation of thrombospondin-1 and thrombospondin-2 after focal cerebral ischemia/reperfusion[END_REF][START_REF] Schachtrup | Fibrinogen inhibits neurite outgrowth via beta 3 integrinmediated phosphorylation of the EGF receptor[END_REF] . TSP1 activates β1 integrins [START_REF] Defreitas | Identification of integrin alpha 3 beta 1 as a neuronal thrombospondin receptor mediating neurite outgrowth[END_REF] and mediates neurite outgrowth and synaptogenesis for functional recovery [START_REF] Liauw | Thrombospondins 1 and 2 are necessary for synaptic plasticity and functional recovery after stroke[END_REF] . By contrast, fibrinogen, a blood protein massively deposited in the spinal cord after injury, inhibits neurite outgrowth via β3 integrins [START_REF] Schachtrup | Fibrinogen inhibits neurite outgrowth via beta 3 integrinmediated phosphorylation of the EGF receptor[END_REF] . Therefore, in addition to their role in maintaining inhibitory synaptic strength under basal conditions, β1 and β3 integrins could modulate glycinergic inhibition in pathological and regenerative situations. These regulations may be crucial in vivo for the modulation of pain pathways [START_REF] Woolf | Neuronal plasticity: increasing the gain in pain[END_REF][START_REF] Harvey | GlyR alpha3: an essential target for spinal PGE2-mediated inflammatory pain sensitization[END_REF] as well as spinal locomotor networks, which mainly rely on the proper equilibrium between glycinergic inhibition and glutamatergic excitation [START_REF] Grillner | The motor infrastructure: from ion channels to neuronal networks[END_REF] . Interestingly, β3 integrins have been shown to regulate the numbers of AMPA receptors at excitatory synapses and are required for synaptic scaling in response to TNF-α [START_REF] Cingolani | Activity-dependent regulation of synaptic AMPA receptor composition and abundance by beta3 integrins[END_REF] . This glia-dependent form of plasticity helps adapt the activity of neuronal networks following chronic activity blockade or sensory deprivation [START_REF] Kaneko | Tumor necrosis factoralpha mediates one component of competitive, experience-dependent plasticity in developing visual cortex[END_REF][START_REF] Stellwagen | Synaptic scaling mediated by glial TNF-alpha[END_REF] .

Altogether, these data and our own suggest that integrins orchestrate inhibitory and excitatory neurotransmission to adjust neuronal excitability in normal and pathological conditions.

Antibodies for immunodetection

α1-GlyRs were immunodetected using either a mouse anti-α1-GlyR (mab2b, 0.7 to 1.25 μg/ml, Synaptic System) or a homemade rabbit anti-α1-GlyR antibody raised against the same extracellular epitope (1:800 to 1:1500). No difference in GlyR lateral dynamics was observed with the mouse and rabbit primary antibodies [START_REF] Levi | Homeostatic regulation of synaptic GlyR numbers driven by lateral diffusion[END_REF] . Other primary antibodies were mouse anti-gephyrin (mAb7a, 1.25 μg/ml, Synaptic System), rabbit anti-gephyrin (homemade, 1:1000), 9E10 (Millipore) and rabbit anti-synapsin I (1.7 μg/ml, Synaptic System). Secondary antibodies used in immunocytochemistry were Cy3-conjugated goat anti-mouse (1.25 μg/ml), Cy3-conjugated goat anti-rabbit (2.5 μg/ml), Alexa 488-conjugated goat anti-rabbit (2.5 μg/ml). Secondary antibodies used in SPT were biotinylated Fab fragments (goat anti-mouse: 1 μg/ml, goat antirabbit: 2 μg/ml). All secondary antibodies were from Jackson ImmunoResearch, except for the Alexa 488-conjugated antibody, which was from Invitrogen.

Immunocytochemistry, image acquisition and quantitative analysis

Immunocytochemistry, image acquisition and fluorescence quantification were performed as previously described [START_REF] Charrier | Cytoskeleton regulation of glycine receptor number at synapses and diffusion in the plasma membrane[END_REF] . Images shown in Supplementary Fig. 2 were obtained using a Leica DM5000B microscope equiped with Yokogawa CSU 10 spinning disk. Sets of neurons compared with quantification were fixed, labeled and imaged in parallel. Analyses were performed using MetaMorph (MetaImaging). GlyR and gephyrin synaptic amounts were quantified in double-labeling experiments by measuring the fluorescence intensity associated with GlyR or gephyrin clusters apposed to presynaptic terminals labeled with synapsin I. In our dissociated spinal cord neurons, 90% of GlyR and gephyrin clusters are apposed to presynaptic terminals [START_REF] Dumoulin | Formation of mixed glycine and GABAergic synapses in cultured spinal cord neurons[END_REF] . Therefore, all GlyR and gephyrin clusters were analyzed when cells were treated with aβ1 and aβ3. Aβ1 and aβ3 effects on GlyR cluster-IR were examined on non-permeabilized neurons. A procedure based on wavelet decomposition was used to quantify synapse density and synapse size (see [START_REF] Hanus | Activity-dependent movements of postsynaptic scaffolds at inhibitory synapses[END_REF] ).

Single particle imaging

Cells were labeled and imaged in MEM recording medium consisting of MEM (Invitrogen) supplemented with 10 mM HEPES, 33 mM glucose, 2 mM glutamine, 1 mM sodium pyruvate and B27 at 37°C. Cells were incubated for 5 min with primary antibodies, washed, incubated for 5 min with biotinylated secondary Fab and washed. Cells were then incubated for 1 min with streptavidin-coated quantum dots emitting at 605 nm (1 nM, Invitrogen) in borate buffer (50 mM). After extensive washing, active presynaptic terminals were stained for 40 s with FM4-64 (3 µM, Invitrogen) in the presence of 40 mM KCl. Cells were imaged in an open chamber mounted on a inverted microscope (IX71, 60X objective, NA=1.45, Olympus). QDs and FM4-64 were detected using a Hg+ lamp and appropriate filters (QD: D455/70x, HQ605/20m; FM4-64: D535/50x, E590lpv2; Chroma Technology). QDs were recorded during 512 consecutive frames at 13 Hz with a CCD camera (Cascade 512BFT; Roper Scientific) and MetaView (MetaImaging). Cells were imaged within 30 min after the labeling.

Single particle tracking and quantitative analysis of lateral diffusion

Analyses were restricted to single QDs identified by the intermittency of their fluorescence. Synapses were identified from FM4-64 images using a procedure based on wavelet decomposition (see [START_REF] Hanus | Activity-dependent movements of postsynaptic scaffolds at inhibitory synapses[END_REF] ).

-Single particle tracking SPT was performed using homemade software written with MATLAB (The MathWorks) [START_REF] Ehrensperger | Multiple association states between glycine receptors and gephyrin identified by SPT analysis[END_REF] . QD localization was determined with a spatial accuracy of about 10 nm by cross correlating the image with a Gaussian model of the point spread function and GlyR-QD trajectories were reconstructed as previously detailed [START_REF] Ehrensperger | Multiple association states between glycine receptors and gephyrin identified by SPT analysis[END_REF] .

-Diffusion coefficients

Diffusion coefficients were calculated from the longest fragment of the trajectory spent in the synaptic and extrasynaptic compartment, determined by comparison of the trajectories with the FM image. Only fragments longer than 30 consecutive frames were taken into account. The mean square displacement (MSD) was calculated using

MSD(ndt) = (N -n) -1 ((x i+n i=1 N -n ∑ -x i ) 2 + (y i+n -y i ) 2
).dt , where x i , y i are the coordinates of an object on frame i, N is the total number of steps in the trajectory, dt is the time interval between two successive frames and ndt is the time interval over which displacement is averaged [START_REF] Saxton | Single-particle tracking: the distribution of diffusion coefficients[END_REF] . Diffusion coefficients (D) were calculated with a fit between data points 2 and 5 of the MSD curves versus time (t) as previously described [START_REF] Ehrensperger | Multiple association states between glycine receptors and gephyrin identified by SPT analysis[END_REF] . Distributions rather than mean values were compared because of the large dispersal of the values (over four orders of magnitude).

Diffusion parameters could vary from one culture another, imposing comparisons with internal controls. Diffusion coefficients at synapses were compared in the 25%-75% inter-quartile range. To evaluate the effects of aβ1 and aβ3 treatments, the distributions were divided into 10 classes with an equal number of values, and the mean diffusion coefficient was calculated for each class. Ratios of the means in aβ1 and aβ3 conditions over the means in control were used as an indicator of aβ1 and aβ3 effects.

-Confinement

Confined trajectories were determined according to the relative deviation of the experimental MSD with the one expected in the case of Brownian diffusion (RD, adapted from [START_REF] Kusumi | Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calciuminduced differentiation in cultured epithelial cells[END_REF] ). The size of the confinement domain for trajectories showing restricted motion was calculated as previously described [START_REF] Ehrensperger | Multiple association states between glycine receptors and gephyrin identified by SPT analysis[END_REF] .

-Dwell time at synapses

The time spent by single GlyR-QDs in the synaptic and extrasynaptic compartments and the number of transitions between the two compartments were determined as previously described [START_REF] Charrier | Cytoskeleton regulation of glycine receptor number at synapses and diffusion in the plasma membrane[END_REF] . A dwell time index was defined as follows: for GlyR-QDs transiting between the synaptic and extrasynaptic compartments, dwell time index = time spent at synapses / (total time detection × n exits from synapses); for GlyR-QDs always detected at synapses, dwell time index = 1. Only GlyR-QDs with a dwell time index > 0.03 were taken into account.

Fluorescence recovery after photobleaching

Experiments were performed at 37°C in MEM recording medium. Active synapses were stained with FM 4-64 as described above. Cells were observed on an inverted microscope (Eclipse TE2000-E, 100X objective, NA=1.4, Nikon) equipped with a DG-4 illumination system (Sutter Instruments) and appropriate filter sets (Semrock, Optoprim), a CCD camera (QuantEM 512SC, Roper Scientific) and a FRAP-3D TM system (Roper Scientific). The procedure was controlled with MetaMorph.

Fluorescence was photobleached using a 488 nm laser at 65 mW for 80 ms in three to four regions of interest (diameter: 1-1.2 μm) centered on individual VeGe synaptic clusters. Photobleaching in the 3-4 regions of interest was quasi-simultaneous.

Fluorescence was monitored in time-lapse. Three images were acquired before the photobleaching within a period of 1 min. The recovery after photobleaching was monitored as follows: 1 image every 10 s for 5 min, 1 image every 20 s for 5 min and 1 image every 60 s for 10 min. At each time point, three images were acquired in zstack with a step size of 0.4 μm. Data were analyzed from maximum intensity projection using MetaMorph. Fluorescence recovery was measured after background subtraction and correction for the ongoing photobleaching as follows: Fcorr t = (F t /F 0 )/(Fnb t /Fnb 0 ), where F t is the fluorescence at time t, F 0 is the mean fluorescence before bleaching, Fnb t is the average fluorescence intensity of three non-bleached spots at time t, and Fnb 0 is the average fluorescence intensity of the same nonbleached spots before bleaching.

Electrophysiology

Whole-cell patch-clamp recordings were performed using a multiclamp 700B controlled by Clampex 10 (Axon Instruments). Patch-pipettes had a tip resistance of The remaining 80% was mixed with neutravidin beads (Pierce) for 2 hrs at 4°C to purify the biotinylated proteins (surface proteins). Proteins were separated and analyzed by SDS-PAGE followed by immunoblotting with mAb4a antibody (Synaptic System, 1:600), rabbit anti-GluR2 antibody (Millipore, 1:200); rabbit anti-GAPDH antibody (Abcam, 1:2,000) and rabbit anti-gephyrin antibody (Santa Cruz, 1:1,500).

Proteins were visualized with enhanced chemiluminescence (ECL+, Amersham). The level of surface receptor was normalized to the corresponding level of total receptor detected. The level of total receptor or gephyrin was normalized to the corresponding level of GAPDH.

In vitro phosphorylation assay

The Ge(4') gephyrin isoform was used to address gephyrin phosphorylation by CaMKII in vitro. This gephyrin isoform was cloned from the rat spinal cord [START_REF] Meier | Functional heterogeneity of gephyrins[END_REF] . It is specifically expressed in the central nervous system [START_REF] Paarmann | Mass spectrometric analysis of glycine receptor-associated gephyrin splice variants[END_REF] and it binds to the GlyR. The C4' cassette of gephyrin does not interfere with GlyR/gephyrin binding in vitro [START_REF] Meier | Functional heterogeneity of gephyrins[END_REF] Autocamtide-2 samples were treated in the same manner.

-Enrichment of gephyrin phosphopeptides Titanium oxide and immobilised metal affinity chromatography pipettes were used for enriching phosphopeptides from the digests. MonoTip TiO pipettes were prepared as per manufacturer's instructions (GL Sciences). Phosphopeptides were bound and washed as previously described [START_REF] Tweedie-Cullen | Comprehensive mapping of posttranslational modifications on synaptic, nuclear, and histone proteins in the adult mouse brain[END_REF] and then eluted twice with 150 μl 0.3 M ammonium hydroxide solution. Unbound peptides were lyophilied and resuspended in 100 μl of binding solution (10% Acetonitrile, 0.1% acetic acid). ZipTip 10 μl Metal Chelator pipette tips (Waters) were prepared and the residual phosphopeptides bound and washed as per manufacturer's instructions (Waters). All phosphopeptide-enriched samples were quickly lowered to pH=3 using TFA and then cleaned with ZipTip C18 tips (Waters) prior to MS analysis.

-MS analysis

Samples were analysed on a calibrated hybrid LTQ Orbitrap XL ETD mass spectrometer (Thermo Scientific). Peptides were resuspended in 3% ACN and 0.2% and then manually evaluated for precise site assignment as previously described [START_REF] Tweedie-Cullen | Comprehensive mapping of posttranslational modifications on synaptic, nuclear, and histone proteins in the adult mouse brain[END_REF] .

Statistics

Data were obtained from at least three independent experiments, except when aβ1 and aβ3 effects were tested after another treatment (2 independent experiments).

  inhibition on the lateral diffusion of GlyRs in the extrasynaptic membrane (n GFX = 675, n GFX+aβ3 = 321, n GFX+aβ1 = 353, statistical tests: NS, Fig. 6c) and at synapses (n GFX = 230, n GFX+aβ3 = 168, n GFX+aβ1 = 218, statistical tests: NS). PKC inhibition also abolished the aβ3-induced increase and the aβ1-induced decrease in the number of GlyRs at synapses (GFX: 100 ± 6%, n = 40; GFX + aβ3: 94 ± 4%, n = 40; GFX + aβ1: 98 ± 5%, n = 40, statistical tests: NS, Fig. 6d). Since integrins regulate GlyR mobility in and out of synapses by different pathways (see above), these results indicate that PKC is necessary for integrin activation but is not responsible for the synapsespecific antagonism between β1 and β3 integrins. Next, we tested the involvement of CaMKII. In the extrasynaptic membrane, inhibition of CaMKII with KN-93 (KN93, 10 μM, 1 hr) had no effect on GlyR mobility (n Ctr = 279, n KN93 = 505, statistical tests: NS, Fig 6e) and did not alter aβ1-and aβ3induced increase of GlyR diffusion coefficients (n KN93+aβ3 = 489, n KN93+aβ1 = 450, p < 0.001). At synapses however, CaMKII inhibition increased GlyR diffusion coefficients (n Ctr = 90, n KN93 = 133, p < 0.001) and decreased their synaptic accumulation (Ctr: 100 ± 6%, n = 40; KN93: 59 ± 3%, n = 40, p < 0.001, Fig. 6f). Furthermore, CaMKII inhibition completely abolished the effects of aβ1 and aβ3 on GlyR mobility at synapses (n KN93+aβ3 = 158, n KN93+aβ1 = 131, statistical tests: NS, Fig. 6e) and on GlyR cluster-IR (KN93: 100 ± 4%, n = 40; KN93 + aβ3: 99 ± 4%, n = 40; KN93 + aβ1: 101 ± 4%, n = 40, statistical tests: NS, Fig. 6f). The effects of aβ1 and aβ3 on gephyrin clusters were also occluded by the CaMKII inhibitor (KN93: 100 ± 4%, n = 40; KN93 + aβ3: 100 ± 5%, n = 40; KN93 + aβ1: 98 ± 4%, n = 40, statistical tests: NS). These experiments reveal the involvement of CaMKII at glycinergic synapses. They indicate that both β1 and β3 integrins signal via CaMKII, which is responsible for their opposing actions at synapses. Furthermore, they demonstrate that controlling GlyR mobility in the synapse is crucial to adjust the number of synaptic GlyRs.

Figure 1

 1 Figure 1 RGD-peptides increase GlyR numbers at synapses. (a) Double immunodetection of α1-GlyR and synapsin I in spinal cord neurons (12 days in vitro) in control or after treatment with echistatin (1 hr, 100 nM). Scale bar: 20 μm. (b) Cumulative probability plot of synaptic GlyR cluster-associated fluorescence in control conditions (black) and after 1-hr echistatin treatment (gray) (n Ctr = 2740; n Echis = 2549; from 21 cells by condition; p < 0.001, MW-test). (c) Increased GlyRassociated fluorescence intensity at synapses after treatment with echistatin or GRGDSP peptide (RGD, gray) compared to control or GRGESP treatment (RGE, black). Mean ± SEM, *** p < 0.001, t-test; a.u.: arbitrary unit.

Figure 2

 2 Figure 2 β1 and β3 integrin blocking antibodies have opposite effects on GlyR numbers at synapses. (a) Immunodetection of α1-GlyR on non-permeabilized cells in control conditions (Ctr) or after 1-hr treatment with β3 (aβ3) or β1 (aβ1) function-blocking antibodies. Scale bar: 20 μm. (b) Normalized fluorescence intensity associated with GlyR clusters (mean ± SEM, *** p < 0.001, t-test). (c) Examples of glycinergic mIPSCs and cumulative probability plot of their amplitudes from 2-min recording periods from the same cells before and 20 min after aβ3 or aβ1 treatment. Scale bars: 100 pA, 1s. (d) Average mIPSC amplitude before and after aβ1 or aβ3 treatment or after a 20-min time lag (Ctr) for each cell. (Ctr: NS; aβ1 and aβ3: p < 0.01, MW-test). (e) Relative effects on mIPSC amplitudes. Mean amplitudes after treatment are plotted relative to the mean amplitudes before treatment (*** p < 0.001, ANOVA). (f) Mean effects on mIPSC frequency (NS, ANOVA). Error bars indicate SEM.

Figure 3

 3 Figure 3 β1 and β3 integrins control GlyR lateral diffusion. (a) Maximum intensity projections of 512 frames recorded at 13 Hz during 38.4 s. The GlyR-QD traces and FM4-64-stained synapses are green and red, respectively; yellow denotes their overlap. The GyR-QD explored area increased after aβ3 or aβ1 treatment in the extrasynaptic membrane (arrowheads). At synapses, aβ3 and aβ1 reduced and increased GlyR-QD mobility (arrows), respectively. Scale bar: 5 μm. (b) Cumulative probability plot of GlyR-QD diffusion coefficients in the extrasynaptic membrane (ES, p < 0.001, MW-test). (c) Cumulative probability plot of GlyR-QD diffusion coefficients at synapses (S, aβ3: p < 0.01, aβ1: p < 0.001, MW-test). (d)

Figure 4

 4 Figure 4 β1 and β3 integrins modulate GlyR confinement and dwell time at synapses. (a) Examples of GlyR-QD trajectories (black) over FM4-64 stained synapses (red) from control (Ctr), aβ3-and aβ1-treated neurons. Trajectories analyzed in b are marked with *. Scale bar: 1 μm. (b-c) Increased and decreased confinement of GlyR-QD after aβ3 (green) and aβ1 (red), respectively. MSD versus time plot (b) for GlyR-QD trajectories indicated in a. Normalized size of the confinement domain at synapses (c). Note that the smaller the confinement domain, the greater the confinement (mean ± SEM, ** p < 0.01, *** p < 0.001, MW-test). (d-e) Dwell time of GlyR-QDs at synapses. Cumulative probability plot (d) and mean dwell time index (e). Error bars indicate SEM; * p < 0.05; *** p < 0.001, MW-test.

Figure 5

 5 Figure 5 β1 and β3 integrins control gephyrin amount and exchanges at synapses. (a) Immunodetection of gephyrin in control conditions (Ctr) or after aβ3 or aβ1 treatment. Scale bar: 20 μm. (b) Normalized fluorescence intensity associated with endogenous gephyrin clusters (mean ± SEM; *** p < 0.001, t-test). (c) Representative example of transfected neurons expressing a Venus-tagged gephyrin (VeGe, green). VeGe forms clusters in front of active presynaptic boutons stained with FM4-64 (red). The white square outlines the region shown in d. Scale bar: 1 μm. (d) Photobleaching of an individual synaptic VeGe cluster (arrow) and time-lapse recording of the fluorescence recovery during 20 min. The color look-up indicates the level of fluorescence. (e) Fluorescence recovery curves (mean ± SEM). (f) Mean fluorescence recovery 10 min after the photobleaching (error bars: SEM, ** p < 0.01; *** p < 0.001, MW-test). F 0 : initial fluorescence.

Figure 6

 6 Figure 6 Actin, PKC and CaMKII mediate integrin-dependent regulation of GlyR lateral dynamics.

  (a, c, e) Cumulative distributions of GlyR-QD diffusion coefficients. (b, d, f) Histograms of normalized fluorescence intensity associated with GlyR clusters (mean ± SEM). (a-b) Effects of aβ1 (red) and aβ3 (green) treatments after F-actin disruption with latrunculin (Lat, 3 μM, black). Latrunculin abolishes extrasynaptic but not synaptic effects on GlyR diffusion and cluster-IR. (c-d) Effects of aβ1 (red) and aβ3 (green) treatments after PKC inhibition (GFX, 50 nM, black). GFX abolishes extrasynaptic and synaptic effects on GlyR diffusion and cluster-IR. (e-f) Effects of CaMKII inhibition (KN93, 10 μM, black) compared with the control (Ctr, blue) and effects of aβ1 (red) and aβ3 (green) treatments after CaMKII inhibition. (e) In the extrasynaptic membrane, KN93 has no effect on GlyR mobility. At synapses, KN93 increases GlyR mobility and prevents aβ1 and aβ3 effects. (f) At synapses, CaMKII inhibition decreases GlyR cluster-IR (blue scale, left) and prevents aβ1 and aβ3 effects on GlyR cluster-IR (black scale, right). *** p < 0.001, ** p < 0.01; for complete statistics, see text.

Figure 7

 7 Figure 7 Thrombospondin 1 and fibrinogen have opposite effects at inhibitory synapses. (a) Typical behavior of GlyR-QDs after 1-hr treatment with thrombospondin 1 (TSP1, 2 μg/ml). Maximum intensity projections of 512 frames recorded at 13 Hz. GlyR-QD explored area: green; FM4-64-stained synapses: red. GlyR-QDs have a reduced mobility in (arrows) and out (arrowheads) of synapses. Scale bar: 1 μm. (b) Distributions of GlyR-QD diffusion coefficients at extrasynaptic and synaptic locations in control condition (Ctr, black) or after TSP1 treatment (red) (p < 0.001, MW-test). (c) Normalized fluorescence intensity associated with GlyR and gephyrin clusters at synapses (mean ± SEM; *** p < 0.001, t-test). (d) Typical behavior of GlyR-QD after 1-hr treatment with fibrinogen (Fib, 1.5 mg/ml). Same legends as for panel a. GlyR-QDs are very mobile at synapses but not in the extrasynaptic membrane. (e) Distributions of GlyR-QD diffusion coefficients at extrasynaptic and synaptic locations after treatment with fibrinogen (green) or with its vehicle (veh, black) (p < 0.001, MWtest). (f) Same legend as for panel c (mean ± SEM; *** p < 0.001, t-test). Note that TSP1 increases the stabilization and the number of GlyRs at synapses whereas fibrinogen has opposite effects.

  formic acid and loaded on a 10 cm fused silica column packed with 3μm 200 Å pore size C18 resin. Peptides were eluted via an ACN gradient of 5-30% ACN over 35 min and 30-80% ACN over the subsequent 13 min in a buffer containing of 0.2% formic acid at flow rate of 200 nl/min. One scan cycle comprised of a full scan MS survey spectrum from m/z 300-2000 acquired in the FT-Orbitrap, followed by up to 6 sequential CID and ETD MS/MS scans with fragment ion detection in the linear ion trap. CID was performed with a target value of 1e4 in the linear trap, collision energy at 35%, Q value at 0.25 and activation time at 30 ms. AGC target values were 5e5 for full FTMS scans and 1e4 for ion trap MSn scans. The ETD anion target value was set at 1e6 and activation time at 100 ms. Supplementary activation was employed to enhance the fragmentation efficiency for 2+ precursors and charge state dependent ETD time enabled. A Data dependent decision tree was used to control ETD dissociation based on charge and m/z. The ETD reaction time was 120 ms and isolation width was 2 m/z. For all experiments, dynamic exclusion was used with 1 repeat count, 30 s repeat duration, and 10 s exclusion duration. Samples were acquired using internal lock mass calibration set on m/z 429.088735 and 445.120025.-Database analysisMS and MS/MS data were processed with Mascot distiller and searched using Mascot version 2.2. Monoisotopic masses of 2+ or greater charged peptides were searched with a peptide tolerance of 8 ppm and an MS/MS tolerance of 0.6 Da for fragment ions using a rat protein database downloaded from the European Bioinformatic Institute. Modifications used for searches included carbamidomethyl (C, variable) and phosphorylation (STY, variable). Only strictly tryptic peptides with a maximum of 4 missed cleavage sites were allowed. Positive identification of phosphorylated peptides was performed using a variety of strict criteria. Only boldred, rank 1 peptides with Mascot expect values of less than 0.05 were considered

Cell surface biotinylation, quantification of GlyR and gephyrin levels

	After 1 hr treatment with aβ1 or aβ3, cells were washed 3 times with ice-cold
	Dulbecco's PBS (DPBS) supplemented with 0.8 mM CaCl 2 and 0.5 mM MgCl 2 , pH
	7.4 (DPBS ++ ). Then, they were incubated twice with biotinylation reagent (1 mg/ml
	NHS-SS-biotin, Pierce) for 12 min with gentle shaking on ice. Cells were then
	washed and unbound biotin was quenched with 50 mM glycine in DPBS ++ /BSA for 15
	min. Cell extracts were collected by scraping the cells in a Tris buffer (TB) consisting
	of 50 mM Tris-HCl (pH=8), 100 mM NaCl, 1 mM PMSF (Sigma) and a protease
	inhibitor cocktail (Roche). Neurons were lysed with 3% Triton X100 in TB
	supplemented with 2 mM EDTA for 1 hr on ice. Cleared Triton extracts were
	collected after centrifugation for 15 min at 10,000 g. The blocking antibodies were
	removed by incubation with protein G immobilized on sepharose (Sigma) in TB
	supplemented with 1% Triton X100 and 2 mM EDTA (Binding buffer, BB) for 1 hr at
	3.5-5 MΩ when filled with an intracellular solution containing 68 mM KGlu, 68 mM
	KCl, 0.2 mM EGTA, 2 mM MgSO 4 , 20 mM HEPES buffer, 3 mM Na 2 ATP, and 0.2
	mM Na 3 GTP. The external solution contained 136 mM NaCl, 2.5 mM KCl, 2 mM
	CaCl 2 , 1.3 mM MgCl 2 , 10 mM HEPES, and 10 mM D-glucose. It was supplemented
	with 1 μM TTX, 50 μM D-AP5, 2 μM NBQX and 5 μM gabazine (SR95531) to isolate
	glycinergic mIPSC. All the supplements were from Tocris. About 10 min after mIPSC
	isolation, aβ1 or aβ3 (20 μg/ml) was added to the external solution for 5 min (flow ≈ 1
	ml/min) to allow the immunoreaction. mIPSCs were recorded at a holding potential of
	-60 mV at 30°C. Recordings were filtered at 4 kHz and sampled at 20 kHz using a
	Digidata 1440A (Axon Instruments). Throughout the experiment, the access
	resistance was tested periodically and cells were discarded if the access resistance
	changed by more than 15% or was more than 20 MΩ. mIPSCs were detected using
	the template procedure of the Clampfit 10 program (Axon Instruments) and analyzed
	over 2-min periods. mIPSCs recorded 15 min after treatment with antibodies were
	compared with mIPSCs before application. In control cells, mIPSCs from 2-min

periods separated by 20 min were compared. All cells with mean mIPSC frequency higher than 1 Hz were taken into account.

4°C. The blocking antibodies were not detected with western blot following this procedure. 20% of the sample was collected to constitute the total receptor fraction.

  Ni-NTA) affinity chromatography as described[START_REF] Bedet | Regulation of gephyrin assembly and glycine receptor synaptic stability[END_REF] . Recombinant CaMKIIα (Cell Signaling Technology) and GST-CaMKIIβ fusion protein (Sigma) were preincubated for 10 min at 30°C in kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 2 mM DTT, 0.1 mM Na 2 EDTA, 200 μM ATP and 10 mM NaF, 2 mM Na 3 VO 4 , 5 mM Na 4 P 2 O 7 .H2O) supplemented with 2 mM CaCl 2 and 1.2 μM calmodulin. In control experiments, CaMKII was preincubated in the absence of CaCl 2 and calmodulin (inactive CaMKII). Recombinant gephyrin Ge(4') was incubated in kinase buffer with activated or inactive CaMKII for 30 min at 30°C. The reaction was stopped by heating at 65°C for 20 min. CaMKII activity was verified using the highly selective CaMKII substrate autocamtide-2 (Sigma).Gephyrin (2 μg) bound to Ni-NTA agarose beads were reduced with 2 mM dithiothreitol for 60 min and alkylated with 4.2 mM iodoacetamide for 1 hr at 21˚C. 100 μl of 100 mM ammonium bicarbonate pH 7.8 and 13 μl MeOH was added and pH adjusted to 7.8. Samples were heated for 15 min at 60˚C prior to the addition of trypsin (1:150 enzyme/substrate). Samples were digested for 3 hr at 37˚C and the reaction stopped by the addition of TFA then lyophilised to remove MeOH.

	Mass Spectrometry
	-On-bead digestion of gephyrin

but it contains a consensus site for phosphorylation by CaMKII. Recombinant Ge(4') has been described previously (Ge(2,4') in reference

[START_REF] Bedet | Regulation of gephyrin assembly and glycine receptor synaptic stability[END_REF]

). Gephyrin was expressed in transformed E. coli and purified under native conditions by nickel-nitrilotriacetic acid (

Acknowledgments

We thank members of the Triller lab; S. Supplisson, K. Aubrey, L. Wang and O. Pascual for kindly providing access to their set-up and for helpful discussions; Y. Goda and L. Cingolani for integrin constructs; C. Specht, B. Barbour and R. Miles for critical reading of the manuscript. This work was supported by INSERM (A.T.), Agence Nationale de la Recherche (ANR08BLAN0282) (A.T.), Fondation Pierre-Gilles de Gennes (A.T.), Institut pour la Recherche sur la Moelle épinière et l'Encéphale (A.T.), Ministère de la Recherche et de la Technologie (C.C. and P.M.), Association Française contre les Myopathies (C.C.). Statistical analyses were performed with StatView (Abacus Concept) or R.

Differences in diffusion coefficients, confinement domain size, dwell time index, FRAP and mIPSCs were assessed using the Mann-Whitney test (MW-test). Data obtained with immunocytochemistry were compared using the unpaired two-tailed Student's t test (t-test). Multiple comparisons of mean effects on diffusion coefficients, mIPSC amplitudes and mIPSC frequency were performed using analysis of variance (ANOVA) followed by the least significant difference post-hoc test.

Normality of the distributions was determined using the one-sample Kolmogorov-Smirnov test. A test was considered significant when p < 0.05. Data are expressed as mean ± SEM.

Figure Legends

Online methods

Primary neuronal culture

Primary cultures of spinal cord neurons were prepared from embryonic Sprague Dawley rats at day 14 as previously described [START_REF] Levi | Strychnine-sensitive stabilization of postsynaptic glycine receptor clusters[END_REF] with few modifications. Cells were plated at a density of 5x10 4 cells/cm 2 . They were maintained in neurobasal medium supplemented with B27 (1X) and L-glutamine (2 mM) at 37°C in 5% CO2 for 11-14 days. The medium was changed every 4-5 days.

Constructs and transfection

Transfections were performed using the Lipofectamine2000 (Invitrogen) method. We used plasmids encoding gephyrin as a fusion protein with Venus yellow fluorescent protein (Vege [START_REF] Hanus | Activity-dependent movements of postsynaptic scaffolds at inhibitory synapses[END_REF] ), EGFP, CTβ1, and CTβ3 (gift from Dr Yukiko Goda, described in [START_REF] Cingolani | Activity-dependent regulation of synaptic AMPA receptor composition and abundance by beta3 integrins[END_REF] ), and c-myc-α1βgb [START_REF] Meier | Formation of glycine receptor clusters and their accumulation at synapses[END_REF] . Cells were transfected 9 or 10 days after plating and imaged 48 hrs (Vege) or 24 hrs (other constructs) later.

Cell treatment

Monoclonal function-blocking antibodies against integrins were hamster anti-rat β1 monoclonal antibody (aβ1, clone Ha2/5) and mouse anti-rat β3 monoclonal antibody (aβ3, clone F11). They were from BD Pharmingen and used at 25 μg/ml unless otherwise noted. Blocking antibodies stabilize the inactive conformation of integrins [START_REF] Hynes | Integrins: bidirectional, allosteric signaling machines[END_REF] .

Echistatin (100 nM), latrunculin A (3 μM), cycloheximide (100 μM) and leupeptin (100 μg/ml) were from Sigma. GRGDSP and GRGESP were from Bachem and used at 200 μM. Purified human platelet TSP1 was from either Sigma or Haematologic Technologies. TSP1 was used at a concentration of 2 μg/ml. Purified human plasma fibrinogen (Calbiochem) was used at 1.5 mg/ml in 1.2 mM sodium citrate buffer. GF109203X/Bisindolylmaleimide I (Calbiochem) was used at 50 nM. KN-93 (10 μM), dynasore (80 μM), MG 132 (50 μM) and PP2 (10 μM) were from Tocris. Unless otherwise noted, cells were treated with aβ1, aβ3, echistatin, TSP1 or fibrinogen for 1 hr before fixation or imaging. Except cycloheximide (2 hrs), inhibitors were added 25

Supplementary Information Titles

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Journal: Nature Neuroscience

Article Title:

A crosstalk between β1 and β3 integrins controls glycine receptor and gephyrin trafficking at inhibitory synapses

Corresponding Author:

Antoine Triller

Supplementary Item & Number

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Title or Caption

Supplementary Figure 1 Effects of aβ1 and aβ3 treatments on synapse density and synapse size and comparison between the effects on GlyR cluster size and fluorescence.

Supplementary Figure 2 Effects of postsynaptic blockade of β1 and β3 integrins on GlyR clusters by overexpression of dominant-negative constructs.

Supplementary Figure 3 Effects of β1 and β3 integrin blocking antibodies on the coefficient of variation of mIPSC amplitudes and on mIPSC kinetics.

Supplementary Figure 4

Aβ1 and aβ3 effects on GlyRs do not depend on endocytosis, exocytosis, protein synthesis or protein degradation.

Supplementary Figure 5

Aβ1 and aβ3 effects on gephyrin do not depend on protein synthesis or protein degradation.

Supplementary Figure 6

Inhibition of Src kinases with PP2 does not prevent the effects of aβ1 and aβ3 on GlyR clusters.

Supplementary Figure 7 PKC inhibition with GF109203X does not affect GlyR lateral dynamics.

Supplementary Figure 8 TSP1 affects GlyR lateral dynamics via β1 integrins whereas fibrinogen activates β3 integrins.

Supplementary Figure 9

Model for the regulation of GlyR lateral dynamics by integrins.

Supplementary Figure 10 CaMKII directly phosphorylates gephyrin in vitro.


F Speziale

THE CIRCULATION OF AYURVEDIC KNOWLEDGE IN INDO-PERSIAN MEDICAL LITERATURE

An important number of texts on Indian medicine were composed in Persian language in India, starting from the Sultanate period (13 th -16 th centuries) and especially during the following Mughal epoch . This can be considered as one of the major movements of scientific translation that took place between various South Asian cultures, as well as the main scientific movement of this kind that took place in the coeval Muslim world. This paper will focus on some of the main features, scholars and texts that characterized this movement of studies. In Mughal India, also Hindu scholars composed Persian scientific texts. Several Hindus studying at the Madrasa, were proficient in Persian and wrote medical and scientific works in this language, some also on the sciences of the Avicennian tradition. Several Persian works on Indian learning were composed for Muslim nobles, and Persian works on Ayurvedic medicine were even dedicated to Awrangzeb (r. 1658-1707). The royal patronage offered in Mughal India to Persian medical works was bigger then that given in contemporary Safavid Persia. However, and in particular in the pharmacological field, these studies were largely stimulated by practical reasons, and the assimilation of the Indian pharmacopoeia acted therefore as a main instrument for the adaptation of the practice of the Muslim physicians to the local conditions.

First of all I would like to specify that I'm not a specialist of Ayurvedic medicine but of Persian medical literature and it is chiefly from the point of view of Persianate studies that I will consider the subject of my talk. I outline in my contribution some of the main features, texts and scholars characterising the movement of writing of Persian texts on Indian medicine that took place in Muslim India. This can be regarded among the important movements of scientific translation that were realised between different South-Asian cultures. Concerning the Muslim world, it represents the main process of transfer of scientific knowledge from a pre-Islamic tradition taking place in the Muslim world during the late medieval and modern epochs. We have recently launched at the IFRI of Tehran a research programme aiming to constitute a first descriptive catalogue of the Persian sources on Indian traditions and sciences. On the basis of a preliminary survey I have made of the existing manuscript sources, it can be said that there exists a corpus of about eighty to one hundred Persian texts on Indian medicine and related subject, such as sexology and veterinary medicine, and including also the important discussions given in Persian texts which are not monographs on Indian medicine.

However, among the translation movements that took place in the Muslim world, the one from Indian sources into Persian remains certainly the lesser know one and many sources remain unknown even in the main bibliographical surveys of Persian literature. One of the main reasons for that lack of interest is the myth of the decline of medical and scientific studies in the Muslim world during the post-medieval period. This view is underlying in most of the general volumes on history of medicine in the Muslim world, being mainly histories of the medieval Arabic texts and authors that were also well known in the European world trough Latin translations. Later sources and authors, as in the case of Ayurvedic tradition, have always been considered as deserving very little attention, since they are essentially sterile repetitions and commentaries of the classic authorities, being the Muslim one a culture where right knowledge was based on the imitation of the past. However, a closer analysis of later medical production, shows surprisingly that many Arabic medical works which were regarded as classics by Western scholars because of their circulation into Latin versions, they were never translated into Persian.

Early contacts between the Muslim and the Indian medical traditions had been already realised during the 9 th century under the Abbaside caliphate of Baghdad. However, it seems that the texts produced in Arabic at this time had a very limited impact on later Muslim medical learning. We do not find any early Arabic text on Indian medicine translated into Persian in Muslim India, not even Rabban al-Tabari section on Indian medicine included in the Firdawas al-hikmat. The Persian texts on Indian medicine produced in India were mainly new compilations. It can be said that Persian studies on Indian medicine had on later Muslim medical culture a much bigger impact than that of earlier Arabic text on the same subject.

To resume a few basic points: the first Indo-Persian works on Indian medicine were composed in medieval India, from the 14 th century onward, under the sultans of Delhi, Gujarat and Malwa. An important development of these studies took place during the Mughal epoch and this trend lasted until the colonial period when works on Indian medicine were written in Urdu as well. During this period, that spread over seven centuries, many Persian works were composed also on other Indian branches of learning and some articles by Carl Ernst have recently analysed in particular the works dealing with yoga and religious traditions. For what concerns the scientific domain field, we find a much bigger number of Persian works on medicine than on other Indian sciences such as mathematics and astronomy. Many dealt in particular with Indian pharmacology; moreover, we find also texts or relevant chapters on Indian sexology, veterinary medicine and iatrochemistry.

It is important to underline some features of the role played by the Persian language as means of scientific expression in India. Mughal India had a much bigger population than Safavid Persia. The Mughal Empire had therefore a bigger Persian speaking population than Safavid Persia and there were many more Persian manuscripts produced in modern India than in Persia. Muslim India was far richer that Persia and Indo-Muslim courts offered a large patronage to Persian speaking scholars and physicians migrating from Iran and Central Asia. In Mughal India, also many Hindu scholars began to study Persian and to compose scientific works in Persian language. Many Hindus, and especially Kayasthas and Khatris, studied at the Madrasa in order to get the munshi (secretary) degree that qualified for administrative posts. The curriculum of the Indian Madrasas included often the study of Avicennian medical and scientific texts, such as the Qanun and its commentaries. Hindus began to write Persian scientific works especially from the second half of the 17 th century. Some important examples are the Rahat al-faras, on the horse and his treatment, by Anand Ram Mukhlis (m. 1164/1751), one of the leading Hindus scholars writing Persian texts and poetry; and the 19 th century Mu'alajat-i hindi by Shaykh Haydar Misri dedicated to the third Nizam of Hyderabad. It is also possible that the early 16 th century Ma'dan al-shifa'-i Sikandar-shahi had been composed with the help of Hindu scholars. The biographies of these Hindu physicians versed in Persian were included in a few collections of biographies of physicians composed in Urdu during the colonial period. During the Mughal epoch, we find even some Christian physicians writing medical texts in Persian, such as those of the family Da Silva. Around the beginning of the 19 th century, José da Silva wrote a Persian treatise on the Indian materia medica, entitled Mufradat-i hindi (Indian simples). However, we have to remark that the most important Persian works on Indian medicine were mainly composed by Muslim physicians and not by Hindu Persian speaking scholars.

We must consider to which extent courtly patronage supported these kinds of studies. Important Persian texts on Indian medicine were dedicated and composed for Muslim nobles. For example, before the Mughal epoch we find already three Persian texts based on the Shalihotra composed for the sultans of Gulbarga, Gujarat and Malwa. We find such texts dedicated also to Muslim sultans that usually are not remembered for a positive attitude towards the Hindus, such as the same Awrangzeb, after whom a few Persian texts on Indian medicine were named. The most important was the Tibb-i Awrangshahi by Darwish Muhammad Imanabadi, a general manual on Indian medicine, which was later copied several times. Of both the other works dedicated to Awrangzeb, the Mirat al-hukama-yi Awrangshahi by Abu al-Fath Chishti and the Dar al-shifa-yi Awrangshahi by Abu al-Fath Khayri, only one existing manuscript is known to us. There is a famous farman by Awrangzeb asking for quicksilver from the abbot of Jakhbar, and extending to him his protection. It is interesting to note that the works by Darwish Muhammad and Abu al-Fath Khayri both included chapters on iatrochemistry. Also if the composition of such texts can be considered as an element of the cultural politic and the imperial ideology of the Mughals which made of Persian translations of Indian works an instrument of this politic, we can say that courtly patronage did not represent the main drive for this movement of studies.

Practical needs, such as finding local substitutes of drugs, played certainly a major role. Indo-Muslim authors explained that they were driven to write such books by the great difficulties encountered in India in identifying and finding the drugs described in Arabic and Persian medieval pharmacopoeias. A colleague has recently drawn my attention to the fact that something similar had happened when the Arabic texts were introduced into Muslim Spain. It was especially in the field of pharmacology and therapeutics that Muslim physicians experimented in India the most important limits of the knowledge transmitted by the Arabic classical works. Pharmacology was the Indian medical discipline that most attracted the interest of the Muslim physicians and it was certainly the field in which the contacts with the local tradition determined the most sensible renewal of the medical knowledge circulating among Indo-Muslim physicians. Indian pharmacology and its lexicon were incorporated as well in several Indo-Persian texts dealing with Avicennian medicine. The integration of the Indian pharmacopoeia acted therefore as a main instrument for the adaptation of the practice of the Muslim physicians to the local conditions.

It seems that the synthesis of Indian knowledge was realised mostly in the practical field, such as pharmacology, and to a much lesser extent in the doctrinal sphere. There are nevertheless a few examples demonstrating that some efforts were made also in this direction. Shihab al-Din Nagawri, a 14 th court physician of the sultan of Gujarat, proposes in his work Tibb-i Shihabi a division of humoral pathology combining the views of the Muslim and the Indian physicians. Shihab al-Din considers the humours as being four, but the two biles, as in the Indian tradition, are regarded as one, and the remaining place is taken by the wind, called bad in Persian. We can observe that this was not something completely new for the Muslim thought, since references to bad are found for example in some medical traditions of the Shiite imams, such as the Risala al-dhahabiyya attributed to the eight imam 'Ali al-Rida (d. 818), of which I've recently made an Italian translation that is in the press.

Persian descriptions of Indian knowledge were adapted through different means to the forma mentis of the Avicennian physicians. Muslim authors employed the Arabic-Persian physiological lexicon to translate and explain the concepts of the Indian doctrine. The Indian drugs were presented according to the typical order, from head to toe, of the nosography of Persian texts. The prescriptions, derived from Indian iatrochemistry, were often included in the class of the elixirs (iksir). The circulation of Indian knowledge in Persian language was made also through its integration in apocryphal texts, such as the Tuhfat al-ashiqin ascribed to Avicenna, and the Haft ahbab, an alchemical text including iatrochemistry, attributed to a group of scholars, among them a couple of Indian Sufis and a Nath yogi converted to Islam. I've given a description of the Haft ahbab based on the manuscript copy preserved in the Library of Leiden, which has been included in the volume Bronnen van Kennis (Leiden, 2006).

It is moreover important to point out that the assimilation of Indian medical knowledge was made also through the reference to the scriptural sources of Islam, and in particular the sayings of prophet Muhammad praising the knowledge of medicine and the quest for scientific knowledge. This kind of discourse was not a completely new one. The compilers of some Medieval Arabic commentaries of the medical traditions of prophet Muhammad were already inclined to show the analogies between Muhammad's traditions and the Greek medical knowledge of Galen. It is interesting to observe that the first noteworthy Indo-Persian medical discussion of the sayings of prophet Muhammad is that given in a treatise on Indian medicine, the Ma'dan al-shifa-yi by Miyan Bhuwa. An important text of this kind is the Mu'alajat-i nabawi (Prophetic remedies) by Ghulam Imam, an author who flourished around the 18 th century. It is a dictionary of drugs dedicated, the author says at the beginning, to the traditions of the prophet Muhammad and the Indian drugs. The prophetic traditions create here the framework of the work and represent an expedient for its composition, but within the text they are mentioned only in reference to a limited number of substances. The reference to the prophetic traditions praising the study of medicine became an element of the rhetorical discourse of the Muslim physicians aiming to present and justify such undertakings according to the Muslim view. Prophetic traditions could act therefore as a powerful symbolic means for the adaptation of the knowledge of the Muslim physicians to local conditions. We can notice moreover that Muslim physicians affiliated to Sufi orders, the main mystical school of Islam, wrote several important Persian works on Indian medicine.

Religious and magical remedies of Indian origin were mentioned in some Persian and Urdu texts, of non-medical contents as well. For instance, the 15th century mystical treatise Lata'if-i Ashrafi by the Sufi Ashraf Jahangir Semnani included Sanskrit mantras against diseases. Both the works on the Indian pharmacopoeia by Aman Allah Khan and by Sharif Khan mention the anti-demoniac properties of several substances. The Mughal emperor Akbar ordered the translation into Persian of the Atharvaveda: however it doesn't seem that this translation was completed.

We must observe an important feature concerning the main kind of Persian texts circulating on Indian medicine. The direct translations of Sanskrit medical works into Persian were not many. The most important Persian works were on the contrary new general manuals on Indian medicine and treatises on Indian pharmacology. Moreover, important Indo-Persian treatises on Avicennian medicine comprised descriptions and discussions of Indian knowledge. It seems therefore that the composition of new works and chapters, and not the translation of previous texts, acted as the principal means for the integration of Indian knowledge into Persian medical literature. Most Persian works on Indian medicine were composed in prose ; nevertheless we find also a few in poetry, such as the Qasida dar lughati hindi (Poem in the Indian Language) by Muhammad ibn Yusuf, a court physician of the Mughals Babur and Humayun and author of other well-known medical texts. Manuscripts copies of some of these texts were illustrated with miniatures, and especially those on sexology and those on the horse and its treatment.

Among the main Persian general treatise on Indian medicine are the Ma'dan al-shifayi Sikandar-shahi by Miyan Bhuwa, the Dastur al-atibba by Firishta and the Tibb Awrangshahi by Darwish Muhammad. Miyan Bhuwa's work was composed in 1512 and was dedicated to the sultan of Delhi, Sikandar Shah Lodhi. During the following century Hasan Muqarrab Khan, a physician and a Mughal nobleman, wrote the 'Ain al-shifa, a treatise on pharmacology based on the Ma'dan al-shifa. The Dastur al-atibba was composed by the Iranian Abu al-Qasim Firishta, the author of a famous history of Muslim India, who lived at the court of the 'Adil Shah sultans of Bijapur during the early 17 th century. In his book Firishta refers to having studied with an Indian physician. The introduction of Dastur alatibba describes the principles of Indian medical doctrine, the first chapter discusses Indian simples and foodstuffs presented in form of a dictionary, the second chapter is on compound drugs, including iatrochemistry, while the last deals with pathology and treatment. The Tibb Awrangshahi by Darwish Muhammad, dedicated to Awrangzeb, is a work divided into seven chapters covering in order: the principles of Ayurveda, anatomy, pathology and therapeutics, woman's diseases, calcinations of metals, purgation, phlebotomy, compound and simple drugs.

Several treatises on the Indian pharmacopoeia were composed during the late Mughal period such as the Mu'alajat-i nabawi by Ghulam Imam, the Anis al-atibba by Nafi' al-Siddiqi al-Ja'isi, the Bustan-i afruz by Sayyid 'Abd al-Fattah, the Mu'alajat-i hindi by Shaykh Haydar Misri, the Muntakhab al-adwiya by Muhammad Qamar al-Din Husayn, and the vast dictionary of Indian simples Mufradat-i hindi by Muhammad Sharf al-Din. The most renowned Persian dictionary of the Indian Materia medica was the Ta'lif-i Sharifi by Muhammad Sharif Khan, a physician who died in the beginning of the 19 th century. Few decades after Sharif Khan's death the text was translated into English by George Playfar, a surgeon on service in Bengal. During the same years was composed in the Deccan the Tadhkirat al-hind, a detailed dictionary of Indian drugs by Riza 'Ali Khan. An earlier version of this book was written in Arabic by 'Ali Khan's father, and stands as one of the rare known texts of this kind composed in Arabic in India.

Indo-Persian works on Avicennian medicine included important descriptions of the Indian knowledge. The two main examples are the 17 th century works by Aman Allah Khan and Nur al-Din Shirazi. The Ganj-i bad-awurd is among the main Indo-Persian works on pharmacology and was composed by Aman Allah Khan, a nobleman of the Mughal court who also translated the Madanavinoda into Persian. The Tibb-i Dara Shikohi by Nur al-Din Shirazi is the biggest Persian medical encyclopaedia written in India and stands as a leading effort aiming to describe in the same book the knowledge of the Muslim and the Indian physicians. The work was dedicated to the Mughal prince Dara Shikoh, one of the most emblematic figures of the contacts between the Muslim and the Indian cultures; his Majma al-bahrayn was the most important Persian treatise offering a comparative description of the philosophic and mystical doctrines of the Muslims and the Hindus.

For what concerns veterinary medicine, this field was characterised by the composition of some monographic works on the horse and the elephant. The early integration of the Indian lexicon of remedies for animals is illustrated by the Tibb-i Firuz-shahi, a 13th century work on the treatment of the falcon employing about fifty Hindustani terms of simples without giving their Persian translation. The Salihotra seems to be the Sanskrit scientific treatise most translated into Persian in India. We find at least seven Persian works, which refer to the Salihotra or have been considered as based on this text. However, a preliminary analysis of some of these Persian versions, such as the 16 th century one by Hashimi (made for the sultan of Gujarat), shows that is rather possible that under this title Muslim physicians did not known only the homonymous work by Shalihotra but also other Sanskrit works having the same title or which were based on the Salihotra. There are several illustrated copies of some Persian works based on the Salihotra, such as some manuscripts of the 17 th century version made for Abd Allah Khan Firuz Jang. This version mentions in the preface the sayings of the Muslim prophets on the horses and a copy preserved in the National Library of Paris includes some miniatures of the horses mentioned in the traditions of the Muslim prophets.

Treatises on the elephant may be regarded as an original contribution of Persian studies. Some of these works on the elephant were certainly based on Indian sources, while for other works we haven't enough information on their contents to say that. A detailed treatise on this subject is the Fil-name by Sa'd Akbar ibn Awliya of which is preserved a manuscript copied in the 19 th century. The author refers to having based his treatise on Brhaspati's Brhaspatimata to which he added some integrations from the work by Kalakapya.

To sum up, I've presented in my talk an introductory description of some of the main features that characterised the movement of Persian studies on Indian medicine. Of course, my aim was not to give a comprehensive vision of this huge corpus of texts, but only to discuss some of its main trends, texts and figures. This corpus of texts shows in particular the limits of some main beliefs of the standard reconstruction of the history of medicine in the modern Muslim world. The Persian studies on the Indian sources did not certainly have an impact on Muslim culture comparable to that which the Greek sources had previously for the early development of Arabic medical studies from the 9 th century. However, this generated a corpus of texts, which in terms of existing sources seems second only to the production that characterised the process of translation from Greek into Arabic. As I said before, I'm not a specialist of Ayurvedic medicine and therefore I hope to benefit a lot from your comments and suggestions to my talk.