Connexin 3 sets synptic strength y controlling stroglil synpse invsion Ulrike Pnnsch, Dominik Freche, Glenn Dllérc, Grégory Ghézli,, Crole Escrtin, Pscl Ezn, Mrtine Cohen-Slmon, Krim Benchenne, Veronic Audr, Amndine Dufour, Jochim H.R. Lüke, Nicole Déglon, Grhm Knott, Dvid Holcmn, Nthlie Rouch, These uthors contriuted eqully to this work Supplementry Figures nd Legends
mepsc frequency (Hz) 1.4 1. 1..8.6.4 ** -5 ** mepscmplitude (pa) - -15-1 -5 Ctrl CNQX Ctrl CNQX Pyrmidl neuron (P16-P5) c Pyrmidl neuron (P8-P1) 1. 1. proility proility Cumultive.8.6.4 1 mipsc frequency (Hz) Cumultive proility.8.6.4 4 6 8 mepsc frequency (Hz) 1. 1..8.6.4 Cumultive proility.8.6.4-5 -1-15 - mipscmplitude (pa) -1 - -3 mepscmplitude (pa) Supplementry Figure 1. Cx3 controls glutmtergic trnsmission of CA1 pyrmidl cells. () Appliction of CNQX (1 nm) for 5 min in hippocmpl slices from Cx3 +/+ mice (n =5 cells) mimics the decrese in mepsc frequency nd mplitude from pyrmidl cells of Cx3 / mice (Frequency, P=, t(4) = 7.16; mplitude P= 65, t(4) = 5.194, pired t test). (,c) Frequency nd mplitude cumultive proility of pyrmidl cell mipscs (n =11 cells for Cx3 +/+ nd Cx3 / mice, ) nd pyrmidl cell mepscs efore onset of Cx3 expression (P8-1 mice, Cx3 +/+ n=11 cells; Cx3 / n = 13 cells, c) re not chnged (mipsc frequency: P=.8186, KS = ; mipsc mplitude: P=.5596, KS = 5; mepsc frequency: P>.9999, KS =.1; mepsc mplitude: P=.391, KS =.3, Kolmogorov-Smirnov). Dt re expressed s men s.e.m. ** P <1
3 3 NeuN S1 Leled cells/field 1 S1 NeuN c Synpse numer/ m 3 1 d PSD re ( m ) 5.15.1.5 e Syn Tu PSD 95 Tu Supplementry Figure. Norml hippocmpl structure in Cx3 / mice. (,) Immunostining of hippocmpl slices for neurons with NeuN nd strocytes with S1 revelsnoltertioninothcelltypesnumersincx3 / mice (n =1 nd 8 fields, respectively) compred to Cx3 +/+ mice (n =1 nd 8 fields, respectively; NeuN: P= 485, t(18) = 1.193; S1: P =.998, t(14) =.1154, unpired t test). Scle r, 4 m for NeuN, 1 m for S1. (c,d) Synpse density in defined volume frctions nd PSD re, nlyzed y EM, re comprle in Cx3 / nd Cx3 +/+ mice (Synpse density: Cx3 +/+ n=6 volume frctions, Cx3 / n=9volumefrctions,p=.851, t(13) = 54, unpired t test; PSD re: Cx3 +/+ n=167 spines; Cx3 / n=56 spines, P =.998, U = 167, Mnn-Whitney). (e) Immunolot nlysis of hippocmpl extrcts from Cx3 +/+ (n=3mice)ndcx3 / mice (n =3 mice) shows no difference in protein expression for synptophysin (Syn) nd PSD-95. Tuulin (Tu). Full-length lots re presented in Supplementry Figure S8d. Dt re expressed s men s.e.m.
8 Normlized EPSC mplitude (%) 15 1 5 1 15 5 3 Time (min) Stimultion intensity ( A) 6 4 5.1.15 5.3 Fier volleymplitude (mv) c threshold seline AHP d -8 15-6 1 15 Resting memrne potentil (mv) -6-4 - Spike mplitude (mv) 1 5 Spike threshold (mv) -4 - AHP (mv) 8 6 4 Numer of spikes 1 5 e EPSC mplitude (% of control) 15 1 5 DPCPX CPP LY3415 Supplementry Figure 3. Cx3 regultes LTP induction, ut does not ffect Schffer collterl fferent responses, intrinsic memrne properties nd excitility of CA1 pyrmidl cells nd gliotrnsmitter relese. () LTP, induced y piring protocol (rrow, Hz stimultion nd depolriztion of the postsynptic cell t mv for 1 min) ypssing insufficient postsynptic ctivtion is norml in CA1 pyrmidl cells from Cx3 / mice (n =4 cells, Cx3 +/+ n=4 cells, P=.3687, t(8) =.955, comprison 15-5 min fter the tetnus, unpired t test). Smple trces represent verged EPSC responses efore nd 15-5 min fter the stimultion. Scle r 1 pa, ms. () Afferent responses of Schffer collterls re unchnged in Cx3 / mice, s ssessed y input-output curves showing presynptic fier volley mplitude s function of stimultion intensity in CA1 strtum rditum of Cx3 / mice (n =1 slices) nd Cx3 +/+ mice (n =9 slices, genotype: P=.936, F(1,1) = 954; stimultion
intensity: P < 1, F(5,1) = 4.88, two-wy ANOVA). (c) Representtive smple trces of ction potentils elicited y current injections (1 pa, 5 ms) in CA1 pyrmidl neurons from Cx3 +/+ nd Cx3 / slices. Scle r, mv, 5 ms. Higher mgnifiction of representtive ction potentil. Scle r, 1 mv, 5 ms. Mesured memrne properties re indicted. (d) Intrinsic memrne properties of CA1 pyrmidl neurons from Cx3 / mice (n =18 cells) re similr to the ones of Cx3 +/+ mice (n = 1 cells, memrne potentil: P=.47, U=88, Mnn-Whitney; spike mplitude: P =.4431, t(8) =.778, unpired t test; spike threshold: P=.145, t(8) = 1.583, unpired t test; AHP: P =.566, U = 94, Mnn-Whitney; numer of spikes: P =.3511, t(8) =.948, unpired t test). Afterhyperpolriztion (AHP). (e) Endogenous ctivtion of denosine-1 receptors (inhiited y DPCPX), NMDA receptors (inhiited y CPP) or metotropic glutmte receptors (inhiited y LY3415) is not chnged in CA1 pyrmidl neurons from Cx3 / mice (DPCPX, n=6 cells; CPP, n=4 cells nd LY3415, n = 4 cells; Cx3 +/+ mice: DPCPX, n = 9 cells, CPP, n = 5 cells, LY3415, n=4 cells; DPCPX: P=.689, U=3; CPP: P=857, U=5; LY3415: P=.6571, U=6, Mnn-Whitney). Dt re expressed s men s.e.m.
1 c d e -1.5-6 -15 * 3 * 3-3 3 Fier volley mplitude (mv) -1. -.5 K current mplitude (pa) -4 - + GLT current mplitude (pa) -1-5 /fier volley fepsp slope 1 1 f Resting memrne potentil (mv) -1-8 -6-4 - Memrne resistnce (M ) 1 8 6 4 g I(nA) 3 V(mV) - -15-1 -5 5 1-1 - -3 Supplementry Figure 4. Cx3 lters synpticlly evoked glutmte trnsporter currents in strocytes without modifying their intrinsic memrne properties. () Simultneous recordings of synpticlly evoked fepsps (smller inset) nd GLT currents in strocytes (1). Kynurenic cid (Kyn, 5 mm) inhiits fepsps (inset) nd reduces the glil potssium current. The remining glil current () consists of fst inwrd current nd slow component of smller mplitude, which cn e isolted y ppliction of TBOA ( M) (3). Sutrction of the slow component isoltes the GLT current (-3). Scle rs,.5 pa, 5 ms for GLT current; mv, ms for fepsp. In hippocmpl slices from Cx3 +/+ (n=1 slices) nd Cx3 / mice (n =1 slices), mplitudes of evoked fier volleys () nd phrmcologiclly isolted potssium currents (c) were similr (: P=.7517, t(18) =.31; c: P=.191, t(11.6) = 1.735, unpired t test with Welch's correction), wheres GLT currents (d) nd fepsp slope/fier volley rtios (e) were ltered (d: P=438, t(18) =.168; e: P=37, t(18) =.5, unpired t test). (f) Resting memrne potentil nd memrne resistnce in strocytes from Cx3 +/+ (n= cells) nd Cx3 / mice (n =16 cells) were similr (Memrne potentil: P=.8917, t(34) =.137, unpired t test; memrne resistnce: P =.3894, U = 13.5, Mnn-Whitney). (g) Current-voltge (I/V) plots of CA1 strocytic currents evoked y 15 ms voltge steps ( 18 4 mv) (Cx3 +/+ n=8 cells, Cx3 / n=5 cells, genotype: P=.6685, F(1,1173) =.1835, voltge step: P < 1, F(,1173) = 415.9, two-wy ANOVA). Scle r, 1 na, 5 ms. Dt re expressed s men s.e.m. * P <5.
Flot-1 Flot-1 c Rft Non-Rft 1 3 4 5 6 7 8 9 1 11 T Rft Non-Rft 1 3 4 5 6 7 8 9 1 11 T -51-51 -51-51 Protein (μg/ l) 6 4 Protein (μg/ l)8 8 6 4 1 3 4 5 6 7 8 9 111 Frction 1 3 4 5 6 7 8 9 111 Frction -.4. -.3-1 fepsp slope (mv ms ) - -.1 Sline Ceftrixone 5.1.15 5.3 Fier volley mplitude (mv) Supplementry Figure 5. Cx3 does not lter the distriution of in rft domins, nd upregultion hs no effect on synptic trnsmission in wild-type mice. (,) Representtive sucrose grdient frctiontion profile for nd flotilin-1 (Flot-1) in Cx3 +/+ () ndcx3 / hippocmpl extrcts (). Full-length lots re presented in Supplementry Figure S8e. Rft domins re recovered in low-density frctions 3-5, which re enriched in flotilin-1, component of lipid rfts. Frctions 8, 9, nd 1 correspond to detergent-solule mteril, nd frction 11 is mde of detergent insolule, non-rft, pellet mteril. An liquot of totl homogente (T) used to lod the discontinuous grdient is lso shown on the sme gel. Protein levels were mesured in ech frction (right). (c) Incresing density in strocytes y ceftrixone tretment does not lter excittory synptic trnsmission in wild-type mice (n = 1 slices) compred to sline treted controls (n =11 slices, tretment: P=.4579, F(1,16) =.5545, fier volley: P < 1 F(5,16) = 56.8, two-wy ANOVA), s investigted y input-output curves. Mice received dily mg/kg ceftrixone or sline for 7 dys, s previously descried 18-. Experiments were performed on dy 7. Dt re expressed s men s.e.m.
15 SR11 d GS Cell som re (μm ) c S1 1 5 S1 e SR11 GFAP Vimentin Merge +kinte f g Stt3 + QUIN & CNTF h CNTF Supplementry Figure 6. Cx3 / strocytes re not hypertrophic or rective. () Hippocmpl strocytes of Cx3 / mice exhiit no difference in cell som size nd overll volume, s shown y the cytoplsmic mrker S1. Scle rs 1 Pm, 5 Pm. () Sulforhodmine 11 (SR11) stining confirmed the S1 stining results. Scle rs 1 Pm, 3 Pm. Som size quntifiction for oth mrkers is shown in (c) (S1: Cx3 / n = 8 cells, Cx3+/+ n = 8 cells, P =.8681, U = 3151, Mnn-Whitney; SR11: Cx3 / n = 3 cells, Cx3+/+ n = cells, P = 11, t(8.34) = 1.8, unpired t test). Immunostining for glutmine synthetse (d), vimentin (e) or signl trnsducer nd ctivtor of trnscription 3 (Stt3) (f) is not different etween Cx3+/+ (n = 3 mice) nd Cx3 / mice (n = 3 mice). As positive control for vimentin expression, hippocmpl sections from Cx3 / mice scrificed 7 dys fter intrperitonel kinte injection (3 mg/kg) were used.
Scle r, 1 m. Positive control for Stt3 leling consists in mice injected in the stritum with lentivirl vector encoding for the cytokine ciliry neurotrophic fctor (CNTF) nd, 4 weeks lter with quinolinte (Quin). Mice were scrificed fter weeks nd disply roust strocyte rectivity in the stritum. Scle r, 3 m. (g,h) Electron microgrphs illustrting representtive strocytic elements in Cx3 / mice (n =3 mice) (g) nd in wild-type mice with CNTF-ctivted strocytes (n =3mice)(h). Note the enhnced filment density in the strocyte () in h. Scle r m. Dt re expressed s men s.e.m.
c s s d PSD Spine Astrocyte Supplementry Figure 7. Seril electron microscopy imges nd three-dimensionl reconstruction of dendritic spine nd surrounding stroglil processes in Cx3 +/+ nd Cx3 / mice. Seril electron microscopy imges from series illustrting n symmetric synpse (xonl outon (), dendritic spine (s)) contcted y stroglil processes (green) in Cx3 +/+ () ndcx3 / mice (c), respectively. Scle r, m. The corresponding reconstructions of the entire dendritic spine (grey) with PSD (red) nd the surrounding strocytic elements (green) re illustrted for Cx3 +/+ () ndcx3 / mice (d), showing closer ssocition of Cx3 / strocytic processes to dendritic spinendshorterdistncetopsd.
Fig.f 8 63 8 63 GLAST Tuulin Fig.g Rft Non-Rft 97 64 51 97 64 51 GLAST Flotilin Rft Non-Rft Rft Non-Rft 64 51 c Fig.3c Cx43 Tuulin Cx6 Tuulin d Supplementry Fig.e PSD-95 Tuulin Synptophysin Tuulin 63 37 37 37 115 8 63 37 37 e Supplementry Fig.5, Rft Non-Rft 1 3 4 5 6 7 8 9 1 11 T 97 64 Rft Non-Rft 1 3 4 5 6 7 8 9 1 11 T Flotilin 51 97 64 Rft Non-Rft 1 3 4 5 6 7 8 9 1 11 T Flotilin 51 Rft Non-Rft 1 3 4 5 6 7 8 9 1 11 T Supplementry Figure 8. Full-length pictures of the lots presented in the min nd supplementry figures. () Figure f. () Figure g. (c) Figure 3c. (d) Supplementry Figure e. (e) Supplementry Figure 5,. Rectngles over the full length gels indicte where the imges for the min nd supplementry figures were cropped.