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• W2464831028 abstract "Research Article27 June 2016Open Access Source DataTransparent process Defective glutamate and K+ clearance by cortical astrocytes in familial hemiplegic migraine type 2 Clizia Capuani Clizia Capuani Department of Biomedical Sciences, University of Padova, Padova, Italy Search for more papers by this author Marcello Melone Marcello Melone Department of Experimental and Clinical Medicine, Università Politecnica delle Marche, Ancona, Italy Center for Neurobiology of Aging, INRCA IRCCS, Ancona, Italy Search for more papers by this author Angelita Tottene Angelita Tottene Department of Biomedical Sciences, University of Padova, Padova, Italy Search for more papers by this author Luca Bragina Luca Bragina Department of Experimental and Clinical Medicine, Università Politecnica delle Marche, Ancona, Italy Center for Neurobiology of Aging, INRCA IRCCS, Ancona, Italy Search for more papers by this author Giovanna Crivellaro Giovanna Crivellaro Department of Biomedical Sciences, University of Padova, Padova, Italy Search for more papers by this author Mirko Santello Mirko Santello orcid.org/0000-0002-7709-4127 Institute of Pharmacology and Toxicology, University of Zurich, Zürich, Switzerland Search for more papers by this author Giorgio Casari Giorgio Casari Vita-Salute San Raffaele University and San Raffaele Scientific Institute, Milano, Italy Search for more papers by this author Fiorenzo Conti Fiorenzo Conti Department of Experimental and Clinical Medicine, Università Politecnica delle Marche, Ancona, Italy Center for Neurobiology of Aging, INRCA IRCCS, Ancona, Italy Fondazione di Medicina Molecolare, Università Politecnica delle Marche, Ancona, Italy Search for more papers by this author Daniela Pietrobon Corresponding Author Daniela Pietrobon orcid.org/0000-0002-5148-8670 Department of Biomedical Sciences, University of Padova, Padova, Italy CNR Institute of Neuroscience, Padova, Italy Search for more papers by this author Clizia Capuani Clizia Capuani Department of Biomedical Sciences, University of Padova, Padova, Italy Search for more papers by this author Marcello Melone Marcello Melone Department of Experimental and Clinical Medicine, Università Politecnica delle Marche, Ancona, Italy Center for Neurobiology of Aging, INRCA IRCCS, Ancona, Italy Search for more papers by this author Angelita Tottene Angelita Tottene Department of Biomedical Sciences, University of Padova, Padova, Italy Search for more papers by this author Luca Bragina Luca Bragina Department of Experimental and Clinical Medicine, Università Politecnica delle Marche, Ancona, Italy Center for Neurobiology of Aging, INRCA IRCCS, Ancona, Italy Search for more papers by this author Giovanna Crivellaro Giovanna Crivellaro Department of Biomedical Sciences, University of Padova, Padova, Italy Search for more papers by this author Mirko Santello Mirko Santello orcid.org/0000-0002-7709-4127 Institute of Pharmacology and Toxicology, University of Zurich, Zürich, Switzerland Search for more papers by this author Giorgio Casari Giorgio Casari Vita-Salute San Raffaele University and San Raffaele Scientific Institute, Milano, Italy Search for more papers by this author Fiorenzo Conti Fiorenzo Conti Department of Experimental and Clinical Medicine, Università Politecnica delle Marche, Ancona, Italy Center for Neurobiology of Aging, INRCA IRCCS, Ancona, Italy Fondazione di Medicina Molecolare, Università Politecnica delle Marche, Ancona, Italy Search for more papers by this author Daniela Pietrobon Corresponding Author Daniela Pietrobon orcid.org/0000-0002-5148-8670 Department of Biomedical Sciences, University of Padova, Padova, Italy CNR Institute of Neuroscience, Padova, Italy Search for more papers by this author Author Information Clizia Capuani1,‡, Marcello Melone2,3,‡, Angelita Tottene1,‡, Luca Bragina2,3, Giovanna Crivellaro1, Mirko Santello4, Giorgio Casari5, Fiorenzo Conti2,3,6 and Daniela Pietrobon 1,7 1Department of Biomedical Sciences, University of Padova, Padova, Italy 2Department of Experimental and Clinical Medicine, Università Politecnica delle Marche, Ancona, Italy 3Center for Neurobiology of Aging, INRCA IRCCS, Ancona, Italy 4Institute of Pharmacology and Toxicology, University of Zurich, Zürich, Switzerland 5Vita-Salute San Raffaele University and San Raffaele Scientific Institute, Milano, Italy 6Fondazione di Medicina Molecolare, Università Politecnica delle Marche, Ancona, Italy 7CNR Institute of Neuroscience, Padova, Italy ‡These authors contributed equally to the work *Corresponding author. Tel: +39 049 827 6052; E-mail: [email protected] EMBO Mol Med (2016)8:967-986https://doi.org/10.15252/emmm.201505944 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Migraine is a common disabling brain disorder. A subtype of migraine with aura (familial hemiplegic migraine type 2: FHM2) is caused by loss-of-function mutations in α2 Na+,K+ ATPase (α2 NKA), an isoform almost exclusively expressed in astrocytes in adult brain. Cortical spreading depression (CSD), the phenomenon that underlies migraine aura and activates migraine headache mechanisms, is facilitated in heterozygous FHM2-knockin mice with reduced expression of α2 NKA. The mechanisms underlying an increased susceptibility to CSD in FHM2 are unknown. Here, we show reduced rates of glutamate and K+ clearance by cortical astrocytes during neuronal activity and reduced density of GLT-1a glutamate transporters in cortical perisynaptic astrocytic processes in heterozygous FHM2-knockin mice, demonstrating key physiological roles of α2 NKA and supporting tight coupling with GLT-1a. Using ceftriaxone treatment of FHM2 mutants and partial inhibition of glutamate transporters in wild-type mice, we obtain evidence that defective glutamate clearance can account for most of the facilitation of CSD initiation in FHM2-knockin mice, pointing to excessive glutamatergic transmission as a key mechanism underlying the vulnerability to CSD ignition in migraine. Synopsis FHM2 is a rare monogenic form of migraine with aura caused by loss-of-function mutations in the astrocytic α2 Na,K ATPase. Investigating the mechanisms underlying the facilitation of cortical spreading depression (CSD) in a genetic mouse model of FHM2 uncovers insights into migraine pathophysiology. The rates of clearance of glutamate and K+ released during cortical activity are both reduced in heterozygous FHM2-knockin mice with 50% reduced expression of the α2 Na,K ATPase (NKA). In FHM2-knockin mice, the membrane density of the glutamate transporter GLT-1 is about 50% reduced in astrocytic processes surrounding cortical excitatory synapses, but is unaltered in axon terminals. The relative impairment of glutamate clearance in FHM2-knockin mice is activity dependent. Most of the facilitation of CSD initiation and a large fraction of the facilitation of CSD propagation in FHM2-knockin mice are due to the defective glutamate clearance by cortical astrocytes. Introduction Migraine is a common disabling brain disease, which manifests itself as recurrent attacks of typically throbbing and unilateral headache with certain associated features such as nausea and hypersensitivity to sensory stimuli. In a third of patients, the headache is preceded by transient focal symptoms that are most frequently visual (migraine with aura: MA). Increasing evidence supports a key role of cortical spreading depression (CSD) in migraine pathogenesis in that CSD underlies migraine aura (Lauritzen, 1994; Noseda & Burstein, 2013; Pietrobon & Moskowitz, 2013, 2014) and may trigger the headache mechanisms (Bolay et al, 2002; Ayata et al, 2006; Zhang et al, 2010, 2011; Karatas et al, 2013; Pietrobon & Moskowitz, 2014; Zhao & Levy, 2015). CSD is a slowly propagating wave of rapid nearly complete depolarization of brain cells lasting about 1 min that silences brain activity for several min (Somjen, 2001; Pietrobon & Moskowitz, 2014). The mechanisms of the primary brain dysfunction underlying the susceptibility to CSD ignition in the human brain and the onset of a migraine attack remain largely unknown and are a major open issue in the neurobiology of migraine. Migraine has a strong multifactorial genetic component (de Vries et al, 2009; Russell & Ducros, 2011; Ferrari et al, 2015). As for many other multifactorial diseases, rare monogenic forms that phenocopy most or all the clinical features of the disorder are helpful for elucidating the molecular and cellular mechanisms of the disease. Familial hemiplegic migraine (FHM) is a rare monogenic autosomal dominant form of MA. Apart from motor features and longer duration of the aura, typical FHM attacks resemble MA attacks and both types of attacks may alternate in patients and co-occur within families (de Vries et al, 2009; Russell & Ducros, 2011; Ferrari et al, 2015). FHM type 2 (FHM2) is caused by mutations in ATP1A2, the gene encoding the α2 subunit of the Na+,K+ ATPase (NKA) (De Fusco et al, 2003; Bøttger et al, 2012). The α2 NKA is expressed primarily in neurons during embryonic development and at the time of birth and almost exclusively in astrocytes in the adult brain (McGrail et al, 1991; Cholet et al, 2002; Ikeda et al, 2003; Moseley et al, 2003). The α2 NKA is thought to play an important role in K+ clearance during neuronal activity, but direct evidence is missing mainly because selective inhibitors allowing to distinguish the contributions of the glial α2 and the neuronal α3 NKA are still lacking (Ransom et al, 2000; D'Ambrosio et al, 2002; Larsen & MacAulay, 2014; Larsen et al, 2014). An important role of α2 NKA in glutamate (Glu) clearance is suggested by its colocalization with the Glu transporters (GluTs) GLT-1 and GLAST in astrocytic processes surrounding glutamatergic synapses in adult somatic sensory cortex (Cholet et al, 2002) and by the association between GluTs and α2 NKA in the same macromolecular complex (Rose et al, 2009). However, this association is not specific for the α2 NKA (Rose et al, 2009; Genda et al, 2011; Illarionava et al, 2014) and, although ouabain pharmacology indicates a preferential functional coupling of GluTs with α2 NKA in cultured astrocytes (Pellerin & Magistretti, 1997; but see Rose et al, 2009; Illarionava et al, 2014), the role of the α2 pump in clearance of synaptically released Glu during neuronal activity remains unclear, given the lack of functional data in brain slices. FHM2 mutations cause the complete or partial loss of function of α2 NKA (De Fusco et al, 2003; Pietrobon, 2007; Tavraz et al, 2008, 2009; Leo et al, 2011; Bøttger et al, 2012; Schack et al, 2012; Swarts et al, 2013; Weigand et al, 2014). α2 NKA protein is barely detectable in the brain of homozygous knockin (KI) mice carrying the pathogenic W887R FHM2 mutation and is halved in the brain of heterozygous W887R/+ mutants (Leo et al, 2011). These FHM2-KI mice show a lower threshold for CSD induction and an increased velocity of CSD propagation, in vivo (Leo et al, 2011), similar to KI mouse models of FHM type 1 (FHM1) (van den Maagdenberg et al, 2004, 2010; Eikermann-Haerter et al, 2009) and to a mouse model of a familial advanced sleep syndrome in which all patients also suffered from MA (Brennan et al, 2013). The mechanisms underlying an increased susceptibility to CSD in FHM2 are unknown, although impaired Glu and/or K+ clearance by astrocytes have been suggested as hypothetical mechanisms (Moskowitz et al, 2004; Pietrobon, 2007). Here, to test these hypotheses and to gain insights into the physiological role of the α2 NKA, we investigated the functional consequences of the W887R FHM2-causing mutation on Glu and K+ clearance by cortical astrocytes during neuronal activity in acute cortical slices. Our findings indicate that the rates of Glu and K+ clearance by cortical astrocytes are reduced in heterozygous W887R/+ KI mice and that the density of GLT-1a in the membrane of astrocytic processes surrounding cortical glutamatergic synapses is reduced by 50% in the FHM2 mutants. Using an in vitro model of CSD and ceftriaxone treatment in FHM2-KI mice as well as pharmacological inhibition of a fraction of GluTs in wild-type (WT) mice, we provide evidence that the defective Glu clearance can account for most of the facilitation of CSD initiation in the FHM2 mouse model. Results To investigate whether the reduced membrane expression of the α2 NKA in heterozygous W887R/+ FHM2-KI mice causes a reduced rate of Glu clearance by cortical astrocytes during neuronal activity, we took advantage of the fact that i) Glu uptake into astrocytes by GluTs is electrogenic, and therefore can be monitored electrophysiologically, and ii) the time course of the Glu transporter current elicited in astrocytes upon extracellular neuronal stimulation in hippocampal slices (the so-called synaptically activated transporter current, STC) reflects, to some extent, the time course of Glu clearance by astrocytes and provides a relative indication of how rapidly synaptically released Glu is taken up from extracellular space (Bergles & Jahr, 1997; Diamond, 2005). The time course of the STC is also affected by the electrotonic properties of the astrocytic membrane, while axon propagation, release asynchrony, glutamate diffusion, and the kinetics of the transporters contribute insignificantly in hippocampal slices (Bergles & Jahr, 1997; Diamond, 2005); thus, the decay kinetics of the STC reflects the rate of Glu clearance filtered by the electrotonic properties of astrocytes (Diamond, 2005). Interestingly, the time course of the STC provides a measure of the rate of Glu clearance that is independent of the amount of Glu released (Diamond & Jahr, 2000; Diamond, 2005; Unichenko et al, 2012). We measured the STC in cortical astrocytes by recording the inward current evoked in layer 1 astrocytes (held at −90 mV, close to the resting potential) by the stimulation of neuronal afferents with an extracellular electrode located in layer 1, 200 μm from the patch-clamped astrocyte, in acute slices of the somatic sensory cortex from P22-23 WT and FHM2-KI mice, in the presence of antagonists of Glu and GABA receptors (Fig 1A) (Bergles & Jahr, 1997; Bernardinelli & Chatton, 2008). The inward current evoked in astrocytes by a single pulse stimulation comprised a rapidly rising and decaying component (complete decay in few tens of ms) and a sustained component (Fig 1B, trace a). The rapidly decaying component was completely inhibited by the GluTs inhibitor TFB-TBOA (TBOA), identifying it as the STC (Fig 1B). The STC can thus be obtained by subtracting the residual current remaining in the presence of TBOA from the total inward current (trace a-b in Fig 1B) (Scimemi & Diamond, 2013). The STC can also be obtained by subtracting from the total inward current an exponential waveform (trace c in Fig 1C) that approximates the average TBOA-insensitive current (Fig 1C, top trace) obtained as described in Materials and Methods (Devaraju et al, 2013; Scimemi & Diamond, 2013). As in hippocampal slices (Diamond & Jahr, 2000; Diamond, 2005), also in barrel cortex slices the decay kinetics of the STC slowed down when the density of GluTs was reduced by subsaturating concentrations of DL-TBOA (Appendix Fig S1A), as expected for a reduced rate of Glu clearance and a longer lifetime of synaptically released Glu in the extracellular space. Moreover, changing the intensity of extracellular stimulation (and hence the number of stimulated fibers and the amount of Glu released) changed the STC amplitude without affecting the STC decay kinetics (Appendix Fig S1B). Thus, to obtain a measure of the rate of clearance of synaptically released Glu by cortical astrocytes, which is not affected by the amount of Glu released by extracellular stimulation, we fitted with an exponential function the decay of the STC (Fig 1). Figure 1. The rate of glutamate clearance by cortical astrocytes, as deduced from the decay kinetics of the synaptically activated glutamate transporter current (STC) elicited by single pulse stimulation, is slower in W887R/+ FHM2-knockin (KI) relative to wild-type (WT) mice Scheme of the STC recording paradigm. The currents elicited by extracellular stimulation in layer 1 were measured in a voltage-clamped layer 1 astrocyte located at 200 μm from the stimulating electrode in an acute slice of mouse barrel cortex. Time constants of decay, τ decay, of the STC isolated pharmacologically in WT and FHM2-KI mice. Top, superimposed representative traces of the inward current evoked in an astrocyte (held at −90 mV) by a single pulse stimulation (indicated by the arrow) in a WT slice, before (trace a) and after (trace b) application of a saturating concentration of the GluT inhibitor TFB-TBOA (TBOA). The STC was obtained by subtracting the residual current remaining in the presence of TBOA from the total inward current (trace a–b); the decay of the STC was best fitted by a single exponential function with τ decay = 6.53 ms (in red). The bar plot shows the average values of τ decay of the STC isolated pharmacologically in cortical slices (n = 13) from P22-23 WT (N = 7) and KI mice (n = 9; N = 3). STC τ decay is 17% higher in KI compared to WT astrocytes (unpaired t-test: **P = 0.003). Hereafter, n indicates the number of slices and N indicates the number of mice. Data are mean ± SEM. τ decay of the STC isolated using an exponential waveform approximating the average TBOA-insensitive current in WT and KI mice. Top trace, average normalized TBOA-insensitive current obtained by pooling the normalized TBOA-insensitive currents recorded in 16 WT and KI cells. This current was best fitted by an exponentially rising function [1-exp (−t/τ rise)] with τ rise = 2.35 ms (in green). The STC was obtained by subtracting from the total current elicited in the astrocyte [a: same representative trace as in (B)] the exponential function A[1-exp (−t/τ rise)] with τ rise = 2.35 ms and A equal to the maximal current measured in the astrocyte at about 60 ms after stimulation (trace c); the decay of the STC (trace a–c) was best fitted by a single exponential function with τ decay = 6.49 ms (in red). The bar plot shows the average values of τ decay of the STC isolated as shown in the top panel in cortical slices from P22-23 WT (n = 28; N = 11) and KI mice (n = 27; N = 9). STC τ decay is 21% higher in KI compared to WT astrocytes (unpaired t-test: ***P < 0.0001). Data are mean ± SEM. Source data are available online for this figure. Source Data for Figure 1 [emmm201505944-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint The STC isolated pharmacologically in cortical astrocytes of WT mice decayed with an exponential time course with time constant τ = 6.80 ± 0.19 ms (Fig 1B). The STC isolated in a larger number of cells using the exponentially rising function that approximates the average TBOA-insensitive current (Fig 1C) decayed with a similar time course (6.46 ± 0.13 ms; unpaired t-test, P = 0.15). The decay kinetics of the STC elicited in cortical astrocytes of heterozygous W887R/+ FHM2-KI mice were slower compared to those in WT astrocytes, as shown by the significantly larger time constant of decay of both the STC isolated pharmacologically (τ = 7.97 ± 0.31 ms) and the STC isolated nonpharmacologically in a larger number of cells (τ = 7.82 ± 0.16 ms) (Fig 1). The slower decay of the STC in FHM2-KI cortical slices indicates that the rate of clearance of synaptically released Glu by cortical astrocytes is slower in FHM2-KI compared to WT mice. The alternative interpretation that the slower decay of the STC in FHM2-KI mice reflects a larger electrotonic filtering in KI compared to WT astrocytes appears quite unlikely, given the similar passive membrane properties measured in KI and WT cortical astrocytes (see Materials and Methods) and the unlikelihood that the astrocyte electrotonic properties are affected by a 50% reduction in α2 NKA expression. Moreover, in the unlikely case that, in contrast with the findings in hippocampal slices (Diamond, 2005), other factors, such as release asynchrony or axonal propagation, contribute significantly to the time course of the STC in cortical slices, the alternative interpretation that a larger contribution of these factors in FHM2-KI mice accounts for the slower decay of the STC in KI slices seems also quite unlikely, given that the FHM2 mutation affects a specific astrocytic NKA that is not expressed in cortical axons (Cholet et al, 2002). We next investigated whether the slowing down of Glu clearance in FHM2-KI mice was larger after repetitive stimulation with trains of 10 pulses at 50 or 100 Hz, as, for example, might be expected if the binding capacity of the GluTs in layer 1 astrocytes is overwhelmed during the train. In this case, the decay kinetics of the STC elicited by the 10th pulse in the train should be slower than those of the STC elicited by a single pulse stimulation (Diamond & Jahr, 2000). Indeed, in experiments in which single stimuli were alternated with trains of 9 and 10 pulses at 50 or 100 Hz, the time constants of decay of the STCs elicited by the 10th pulse of the high-frequency trains (isolated as described in Materials and Methods: Fig 2A) were significantly larger than those of the corresponding STC elicited by a single pulse. For example, the time constant of the STC elicited in WT astrocytes by the 10th pulse of a 50-Hz train, τ 10 (50 Hz), was 7.56 ± 0.16 ms (n = 23), while that elicited by a single pulse in the same cells was τ 1 = 6.45 ± 0.15 ms (n = 23; paired t-test, P < 0.0001) and the average ratio τ 10/τ 1 (50 Hz) was 1.18 ± 0.02 (n = 23). The slowing of the STC after the train was larger in FHM2-KI compared to WT mice (in KI: τ 10/τ 1 (50 Hz) = 1.26 ± 0.03, n = 21; unpaired t-test, P = 0.021). The relative slowing and the difference between WT and KI mice were more pronounced after a 100-Hz train (τ 10/τ 1 (100 Hz) = 1.25 ± 0.03, n = 18, in WT versus τ 10/τ 1 (100 Hz) = 1.40 ± 0.05, n = 14, in KI, P = 0.009). As a consequence, the slowing down of Glu clearance in FHM2-KI compared to WT mice was quantitatively larger after repetitive stimulation than after single pulse stimulation and increased with increasing stimulation frequency (Fig 2B). The time constants of decay of the STCs elicited by the 10th pulse of 50- and 100-Hz trains in FHM2-KI mice (τ 10(50 Hz) = 9.82 ± 0.24; τ 10(100 Hz) = 11.08 ± 0.41 ms) were 30 and 37% larger than those in WT mice, respectively (τ 10(50 Hz) = 7.56 ± 0.16 ms; τ 10(100 Hz) = 8.09 ± 0.23 ms) (Fig 2B). For comparison, the time constant of decay of the STC elicited by a single pulse was 21% larger in FHM2-KI compared to WT mice (Fig 1). Figure 2. The slowing down of glutamate clearance in FHM2-KI relative to WT mice is larger after a train of action potentials at high frequency than after a single action potential Isolation of the STC elicited by the last pulse of a high-frequency train of action potentials. Left: superimposed representative traces of the inward current evoked in an astrocyte (held at −90 mV) by extracellular stimulation with a train of 10 pulses (trace a: black) and a train of nine pulses (trace b: brown) at 50 Hz in a WT cortical slice. The inward current elicited by the 10th pulse was obtained by subtracting the current elicited by nine pulses from that elicited by 10 pulses (trace a-b). Right: The STC elicited by the 10th pulse (trace a-b-c) was obtained by subtracting the exponential function that simulates the TBOA-insensitive current elicited by a single pulse (trace c, obtained as in Fig 1C) to the 10-9 pulses difference current (trace a-b). The decay of the STC elicited by the 10th pulse was best fitted by a single exponential function with τ decay = 8.04 ms (in red). τ decay of the STC elicited by the 10th pulse of 50-Hz (τ 10 (50 Hz), left panel) and 100-Hz (τ 10 (100 Hz), right panel) trains in layer 1 astrocytes in acute cortical slices from P22-23 WT (n = 23; N = 10 for 50 Hz; n = 18; N = 8 for 100 Hz) and KI (n = 21; N = 9 for 50 Hz and n = 14; N = 7 for 100 Hz) mice. The STC elicited by the 10th pulse was obtained as described in (A). STC τ 10 (50 Hz) and τ 10 (100 Hz) are 30% and 37% higher in KI compared to WT mice, respectively (unpaired t-test: ***P < 0.0001 in both cases). Data are mean ± SEM. Source data are available online for this figure. Source Data for Figure 2 [emmm201505944-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint Given the evidence of association in the same protein complex of the glial GluTs (GLT-1 and GLAST) and the α2 NKA pump (Rose et al, 2009; Illarionava et al, 2014) and of colocalization of the glial GluTs with α2 NKA in astrocytic processes surrounding neocortical glutamatergic synapses (Cholet et al, 2002), we investigated whether the reduced expression of α2 NKA leads to a reduced density of GLT-1 in the membrane of astrocytic processes surrounding cortical excitatory synapses (perisynaptic astrocytic processes: PAPs). GLT-1 is the quantitatively dominant GluT in the brain and mediates the majority of Glu clearance in the adult murine neocortex (Haugeto et al, 1996; Rothstein et al, 1996; Tanaka et al, 1997; Danbolt, 2001; Campbell et al, 2014). We obtained a first indication that GLT-1 expression in the vicinity of cortical glutamatergic synapses was decreased in FHM2-KI mice from double-labeling immunofluorescence of GLT-1a and the vesicular glutamate transporter VGLUT1 in neocortical sections. GLT-1a is the predominant brain GLT-1 isoform (Chen et al, 2004; Berger et al, 2005; Holmseth et al, 2009), and VGLUT1 is expressed in the large majority of cortical excitatory terminals (Kaneko et al, 2002). Quantitative analysis of GLT-1a immunoreactivity (ir) showed that the mean size of the GLT-1a-positive (GLT-1a+) ir puncta (green) that overlapped with VGLUT1+ ir puncta (red) was reduced by 18% in FHM2-KI mice (0.47 ± 0.03 μm2) compared to WT (0.57 ± 0.03 μm2) (Fig 3). The percentages of GLT-1a+ puncta overlapping with VGLUT1+ puncta were comparable (49 ± 3% in KI; 46 ± 4% in WT). As a change in size of ir puncta has been considered strongly suggestive of a change in protein expression (Bozdagi et al, 2000; Bragina et al, 2006; Omrani et al, 2009), the reduced size of the GLT-1a+ puncta overlapping with VGLUT1+ puncta is consistent with a reduced GLT-1a expression in the vicinity of cortical glutamatergic synapses in W887R/+ KI mice. Figure 3. The size of GLT-1a-immunoreactive (ir) puncta overlapping with VGLUT1 ir puncta is reduced in the cortex of W887R/+ FHM2-KI mice, suggesting a reduced expression of the glutamate transporter GLT-1a in the vicinity of cortical glutamatergic synapses Simultaneous visualization of GLT-1a+ (green) and VGLUT1+ puncta (red) in first somatic sensory cortex (SI) of a WT and a KI mouse (P35). Arrows point to some GLT-1a+ puncta overlaying with VGLUT1+ puncta (i.e. GLT-1a/VGLUT1-related puncta); framed regions (enlarged below) are examples of GLT-1a/VGLUT1-related puncta (arrowheads). All microscopic fields are from layers II/III. Scale bar: 3.5 μm for left and right upper panels and 1 μm for enlarged framed areas. Percentage and size of GLT-1a+ puncta overlaying with VGLUT1 in P35 WT and KI mice. Left, percentage of GLT-1a+ puncta overlaying with VGLUT1 is comparable in WT and KI mice (data were obtained from 14 and 18 fields of 20 × 20 μm from 2 WT and 2 KI mice (four sections/animal), respectively) (unpaired t-test: P = 0.52). Right, size of GLT-1a/VGLUT1-related puncta is 18% reduced in KI mice (169 GLT-1a+ puncta analyzed from 2 mice) compared to WT (174 GLT-1a+ puncta analyzed from 2 mice) (Mann–Whitney U-test: *P = 0.016). Data are mean ± SEM. Source data are available online for this figure. Source Data for Figure 3 [emmm201505944-sup-0005-SDataFig3.pdf] Download figure Download PowerPoint The majority of overlapping GLT-1a/VGLUT1 puncta colocalized with α2 NKA (81 ± 2%), as revealed by triple-labeling immunofluorescence of GLT-1a, VGLUT1, and α2 NKA in WT cortical sections (Fig EV1). This suggests a strict colocalization of GLT-1a and α2 NKA in the astrocytic processes close to glutamatergic synapses, given that in the adult brain α2 NKA is expressed almost exclusively in astrocytes and is not present in cortical axon terminals (McGrail et al, 1991; Cholet et al, 2002). Click here to expand this figure. Figure EV1. Colocalization of GLT-1a and α2 NKA in the vicinity of cortical glutamatergic synapses in WT miceSimultaneous visualization of GLT-1a (green), α2 Na+,K+ ATPase (α2 NKA) (red), and VGLUT1 (blue) immunoreactivity puncta in a section of first somatic sensory cortex (SI) of a WT mouse. Confocal microscopy inspection of fields reveals a high degree of colocalization between GLT-1a and α2 NKA in GLT-1a+ puncta overlaying with VGLUT1+ puncta (arrows). Framed regions (enlarged below) show examples of overlapping GLT-1a/VGLUT1 puncta that are colocalized with α2 NKA. Data obtained from 32 fields of 20 × 20 μm from 2 P32 WT mice (3 sections/animal) revealed that 81 ± 2% of overlapping GLT-1a/VGLUT1 puncta colocalized with α2. Scale bar: 3.5 μm for upper panel and 1 μm for enlarged framed areas. All microscopic fields are from layers II/III. Source data are available online for this figure. Download figure Download PowerPoint We then used post-embedding immunogold" @default.
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• W2464831028 date "2016-06-27" @default.
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• W2464831028 title "Defective glutamate and K ⁺ clearance by cortical astrocytes in familial hemiplegic migraine type 2" @default.
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