Astrocytes play active roles in brain physiology by dynamic interactions with neurons. Connexin 30, one of the two main astroglial gap-junction subunits, is thought to be involved in behavioral and basic cognitive processes. However, the underlying cellular and molecular mechanisms are unknown. We show here in mice that connexin 30 controls hippocampal excitatory synaptic transmission through modulation of astroglial glutamate transport, which directly alters synaptic glutamate levels. Unexpectedly, we found that connexin 30 regulated cell adhesion and migration and that connexin 30 modulation of glutamate transport, occurring independently of its channel function, was mediated by morphological changes controlling insertion of astroglial processes into synaptic clefts. By setting excitatory synaptic strength, connexin 30 plays an important role in long-term synaptic plasticity and in hippocampus-based contextual memory. Taken together, these results establish connexin 30 as a critical regulator of synaptic strength by controlling the synaptic location of astroglial processes.
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We thank R. Nicoll, A. Koulakoff, E. Brouillet, O. Chever, F. Mammano and D. Schmitz for discussions and for technical assistance. This work was supported by grants from the Human Frontier Science Program Organization (Career Development Award), French Research Agency (Programme Jeunes chercheurs and Programme Blanc), City of Paris (Programme Emergence) and INSERM to N.R., from CEA and CNRS to C.E. and N.D., from International Brain Research Organization to V.A., from the French Research Ministry and Deutsche Forschungsgemeinschaft to U.P., from Labex Memolife to G.D., from the doctoral school ED3C, Paris 6 University to G.G. and from the Max-Planck Society to D.F.
The authors declare no competing financial interests.
Integrated supplementary information
(a) Application of CNQX (100 nM) for 5 min in hippocampal slices from Cx30+/+ mice (n = 5 cells) mimics the decrease in mEPSC frequency and amplitude from pyramidal cells of Cx30−/− mice (Frequency, P = 0.0020, t(4) = 7.162; amplitude P= 0.0065, t(4) = 5.194, paired t test). (b,c) Frequency and amplitude cumulative probability of pyramidal cell mIPSCs (n = 11 cells for Cx30+/+ and Cx30−/− mice, b) and pyramidal cell mEPSCs before onset of Cx30 expression (P8-10 mice, Cx30+/+ n = 11 cells; Cx30−/− n = 13 cells, c) are not changed (mIPSC frequency: P = 0.8186, KS = 2; mIPSC amplitude: P = 0.5596, KS = 0.25; mEPSC frequency: P> 0.9999, KS = 0.1000; mEPSC amplitude: P = 0.3291, KS = 0.3, Kolmogorov-Smirnov). Data are expressed as mean ±s.e.m. ** P < 0.01.
(a,b) Immunostaining of hippocampal slices for neurons with NeuN and astrocytes with S100 reveals no alteration in both cell types numbers in Cx30−/− mice (n = 10 and 8 fields, respectively) compared to Cx30+/+ mice (n = 10 and 8 fields, respectively; NeuN: P = 0.2485, t(18) = 1.193; S100: P = 0.9098, t(14) = 0.1154, unpaired t test). Scale bar, 400 μm for NeuN, 100 μm for S100. (c,d) Synapse density in defined volume fractions and PSD area, analyzed by EM, are comparable in Cx30−/− and Cx30+/+ mice (Synapse density: Cx30+/+ n = 6 volume fractions, Cx30−/− n = 9 volume fractions, P = 0.8251, t(13) = 0.2254, unpaired t test; PSD area: Cx30+/+ n = 167 spines; Cx30−/− n = 256 spines, P = 0.9298, U = 21607, Mann-Whitney). (e) Immunoblot analysis of hippocampal extracts from Cx30+/+ (n = 3 mice) and Cx30−/− mice (n = 3 mice) shows no difference in protein expression for synaptophysin (Syn) and PSD-95. Tubulin (Tub). Full-length blots are presented in Supplementary Figure S8d. Data are expressed as mean ±s.e.m.
Supplementary Figure 3 Cx30 regulates LTP induction, but does not affect Schaffer collateral afferent responses, intrinsic membrane properties and excitability of CA1 pyramidal cells and gliotransmitter release.
(a) LTP, induced by a pairing protocol (arrow, 2 Hz stimulation and depolarization of the postsynaptic cell at 0 mV for 1 min) bypassing insufficient postsynaptic activation is normal in CA1 pyramidal cells from Cx30−/− mice (n = 4 cells, Cx30+/+ n = 4 cells, P = 0.3687, t(8) = 0.9525, comparison 15-25 min after the tetanus, unpaired t test). Sample traces represent averaged EPSC responses before and 15-25 min after the stimulation. Scale bar 10 pA, 20 ms. (b) Afferent responses of Schaffer collaterals are unchanged in Cx30−/− mice, as assessed by input-output curves showing presynaptic fiber volley amplitude as a function of stimulation intensity in CA1 stratum radiatum of Cx30−/− mice (n = 10 slices) and Cx30+/+ mice (n = 9 slices, genotype: P = 0.9236, F(1,102) = 0.009254; stimulation intensity: P < 0.0001, F(5,102) = 40.88, two-way ANOVA). (c) Representative sample traces of action potentials elicited by current injections (100 pA, 500 ms) in CA1 pyramidal neurons from Cx30+/+ and Cx30−/− slices. Scale bar, 20 mV, 50 ms. Higher magnification of a representative action potential. Scale bar, 10 mV, 5 ms. Measured membrane properties are indicated. (d) Intrinsic membrane properties of CA1 pyramidal neurons from Cx30−/− mice (n = 18 cells) are similar to the ones of Cx30+/+ mice (n = 12 cells, membrane potential: P = 0.4072, U = 88, Mann-Whitney; spike amplitude: P = 0.4431, t(28) = 0.7780, unpaired t test; spike threshold: P = 0.1245, t(28) = 1.583, unpaired t test; AHP: P = 0.5660, U = 94, Mann-Whitney; number of spikes: P = 0.3511, t(28) = 0.9482, unpaired t test). Afterhyperpolarization (AHP). (e) Endogenous activation of adenosine-1 receptors (inhibited by DPCPX), NMDA receptors (inhibited by CPP) or metabotropic glutamate receptors (inhibited by LY341495) is not changed in CA1 pyramidal neurons from Cx30−/− mice (DPCPX, n = 6 cells; CPP, n = 4 cells and LY341495, n = 4 cells; Cx30+/+ mice: DPCPX, n = 9 cells, CPP, n = 5 cells, LY341495, n = 4 cells; DPCPX: P = 0.6809, U = 23; CPP: P = 0.2857, U = 5; LY341495: P = 0.6571, U = 6, Mann-Whitney). Data are expressed as mean ±s.e.m.
Supplementary Figure 4 Cx30 alters synaptically evoked glutamate transporter currents in astrocytes without modifying their intrinsic membrane properties.
(a) Simultaneous recordings of synaptically evoked fEPSPs (smaller inset) and GLT currents in astrocytes (1). Kynurenic acid (Kyn, 5 mM) inhibits fEPSPs (inset) and reduces the glial potassium current. The remaining glial current (2) consists of a fast inward current and a slow component of smaller amplitude, which can be isolated by application of TBOA (200 μM) (3). Subtraction of the slow component isolates the GLT current (2-3). Scale bars, 2.5 pA, 25 ms for GLT current; 0.2 mV, 2 ms for fEPSP. In hippocampal slices from Cx30+/+ (n = 10 slices) and Cx30−/− mice (n = 10 slices), amplitudes of evoked fiber volleys (b) and pharmacologically isolated potassium currents (c) were similar (b: P = 0.7517, t(18) = 0.3212; c: P = 0.1091, t(11.6) = 1.735, unpaired t test with Welch's correction), whereas GLT currents (d) and fEPSP slope/fiber volley ratios (e) were altered (d: P = 0.0438, t(18) = 2.168; e: P = 0.0372, t(18) = 2.25, unpaired t test). (f) Resting membrane potential and membrane resistance in astrocytes from Cx30+/+ (n = 20 cells) and Cx30−/− mice (n = 16 cells) were similar (Membrane potential: P = 0.8917, t(34) = 0.1372, unpaired t test; membrane resistance: P = 0.3894, U = 132.5, Mann-Whitney). (g) Current-voltage (I/V) plots of CA1 astrocytic currents evoked by 150 ms voltage steps (+180 – +40 mV) (Cx30+/+ n = 28 cells, Cx30−/− n = 25 cells, genotype: P = 0.6685, F(1,1173) = 0.1835, voltage step: P < 0.0001, F(22,1173) = 415.9, two-way ANOVA). Scale bar, 1 nA, 25 ms. Data are expressed as mean ± s.e.m. * P < 0.05.
Supplementary Figure 5 Cx30 does not alter the distribution of GLT1 in raft domains, and GLT1 upregulation has no effect on synaptic transmission in wild-type mice.
(a,b) Representative sucrose gradient fractionation profile for GLT1 and flotilin-1 (Flot-1) in Cx30+/+ (a) and Cx30−/− hippocampal extracts (b). Full-length blots are presented in Supplementary Figure S8e. Raft domains are recovered in low-density fractions 3-5, which are enriched in flotilin-1, a component of lipid rafts. Fractions 8, 9, and 10 correspond to detergent-soluble material, and fraction 11 is made of detergent insoluble, non-raft, pellet material. An aliquot of total homogenate (T) used to load the discontinuous gradient is also shown on the same gel. Protein levels were measured in each fraction (right). (c) Increasing GLT1 density in astrocytes by ceftriaxone treatment does not alter excitatory synaptic transmission in wild-type mice (n = 12 slices) compared to saline treated controls (n = 11 slices, treatment: P = 0.4579, F(1,126) = 0.5545, fiber volley: P < 0.0001 F(5,126) = 56.80, two-way ANOVA), as investigated by input-output curves. Mice received daily 200 mg/kg ceftriaxone or saline for 7 days, as previously described18-20. Experiments were performed on day 7. Data are expressed as mean ± s.e.m.
Hippocampal astrocytes of Cx30−/− mice exhibit no difference in cell soma size and overall volume, as shown by the cytoplasmic marker S100. Scale bars 10 μm, 50 μm. (b) Sulforhodamine 101 (SR101) staining confirmed the S100 staining results. Scale bars 10 μm, 30 μm. Soma size quantification for both markers is shown in (c) (S100: Cx30−/− n = 80 cells, Cx30+/+ n = 80 cells, P = 0.8681, U = 3151, Mann-Whitney; SR101: Cx30−/− n = 32 cells, Cx30+/+ n = 20 cells, P = 0.2110, t(28.34) = 1.280, unpaired t test). Immunostaining for glutamine synthetase (d), vimentin (e) or signal transducer and activator of transcription 3 (Stat3) (f) is not different between Cx30+/+ (n = 3 mice) and Cx30−/− mice (n = 3 mice). As a positive control for vimentin expression, hippocampal sections from Cx30−/− mice sacrificed 7 days after intraperitoneal kainate injection (30 mg/kg) were used. Scale bar, 10 μm. Positive control for Stat3 labeling consists in mice injected in the striatum with a lentiviral vector encoding for the cytokine ciliary neurotrophic factor (CNTF) and, 4 weeks later with quinolinate (Quin). Mice were sacrificed after 2 weeks and display robust astrocyte reactivity in the striatum. Scale bar, 30 μm. (g,h) Electron micrographs illustrating representative astrocytic elements in Cx30−/− mice (n = 3 mice) (g) and in wild-type mice with CNTF-activated astrocytes (n = 3 mice) (h). Note the enhanced filament density in the astrocyte (a) in h. Scale bar 0.2 μm. Data are expressed as mean ±s.e.m.
Supplementary Figure 7 Serial electron microscopy images and three-dimensional reconstruction of a dendritic spine and surrounding astroglial processes in Cx30+/+ and Cx30−/− mice.
Serial electron microscopy images from a series illustrating an asymmetric synapse (axonal bouton (b), dendritic spine (s)) contacted by astroglial processes (green) in Cx30+/+ (a) and Cx30−/− mice (c), respectively. Scale bar, 0.2 μm. The corresponding reconstructions of the entire dendritic spine (grey) with PSD (red) and the surrounding astrocytic elements (green) are illustrated for Cx30+/+ (b) and Cx30−/− mice (d), showing a closer association of Cx30−/− astrocytic processes to dendritic spine and a shorter distance to PSD.
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Pannasch, U., Freche, D., Dallérac, G. et al. Connexin 30 sets synaptic strength by controlling astroglial synapse invasion. Nat Neurosci 17, 549–558 (2014). https://doi.org/10.1038/nn.3662
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