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Discipline
Biological
Keywords
EPS8
Synapse
ASD
Environmental Enrichment
Magnesium
Observation Type
Standalone
Nature
Standard Data
Submitted
Jan 10th, 2019
Published
Mar 28th, 2019
  • Abstract

    Several neuropsychiatric diseases, including autism spectrum disorders (ASD) and intellectual disability (ID) are characterized by synaptic dysfunctions, such as, altered number and shape of dendritic spines, and defective synaptic signalling and plasticity. The actin regulatory protein, Eps8, plays a key role in spine morphogenesis and plasticity since neurons lacking Eps8 show defective spine morphology and density and fail to undergo long term potentiation. Consistently, Eps8 KO mice display increased density of immature spines in CA1 hippocampal region and cognitive defects. Also, lower levels of Eps8 have been detected in the brain of a cohort of ASD patients. Preclinical studies showed that environmental factors, such as mice exposure to environmental enrichment (EE) or neuron exposure to increased extracellular Mg2+, may have a “plasticizing action” resulting in improved cognitive functions and higher cellular plasticity. Here, we tested whether early EE or high Mg2+ exposure may rescue the structural synaptic defects of Eps8 KO mice and neurons. Results of this study indicate that the early EE treatment in mice and elevated extracellular Mg2+ concentration in neurons restore normal dendritic morphology and synaptic plasticity in Eps8 KO mice and neurons. These data suggest that early “plasticizing” interventions can be beneficial on synaptic defects and their efficacy in the treatment of neuro-developmental diseases is worth to be investigated.

  • Figure
  • Introduction

    Several neuropsychiatric diseases, including autism spectrum disorders (ASD) and intellectual disability (ID) are characterized by synaptic pathology, consisting in abnormal density and morphology of dendritic spines, synapse loss and aberrant synaptic signalling and plasticity. For these reasons they are collectively called synaptopathies. Of relevance, the dendritic spine dysgenesis found in individuals with autism related disorders is consistently replicated in experimental mouse models.

    Dendritic spines are small actin-rich protrusions from neuronal dendrites that form the postsynaptic compartment of most excitatory synapses and are essential sites for learning and memory. A tight regulation of the actin cytoskeleton is essential to the formation, maturation, and plasticity of dendritic spines. Among the molecular players involved in this process, Eps8, a multi-functional actin-binding protein that regulates actin remodelling through Rac modulation and actin capping and bundling activities, has been found to play a key role in spine morphogenesis and plasticity. Eps8 is recruited to the spine head during LTP and inhibition of its capping activity impairs spine enlargement and plasticity. Cultured neurons lacking Eps8 show an abnormal increased density of spines which display an immature filopodia-like shape and are unable to undergo morphological and functional synaptic potentiation upon chemical-LTP protocol. Also altered NMDA receptors composition has been found in Eps8 KO neurons. Consistently, Eps8 knock out mice (Eps8 KO) display increased density of immature spines in CA1 hippocampal region and are impaired in cognitive functions. Of note reduced Eps8 levels have been detected in the brain of post-mortem ASD patients.

    Environmental enrichment, which provides enhanced sensory, cognitive and physical activity in rearing conditions has been shown to improve cognitive functions, cellular plasticity and associated molecular processes in mouse models of brain diseases. Also, it has been proposed as an effective treatment for ASD. Indeed, the behavioural therapy – such as applied behavioural analysis (ABA) - is usually the first-line treatment of children affected by ASD that experience difficulty in developing social, speech, and behavioural skills, while the pharmacological therapies help patients function in their daily activities. In particular, risperidone and aripiprazole, which remain the pillars of ASD treatment, are FDA-approved drugs only for managing the irritability associated with the disorder.

  • Objective

    Based on these premises, we investigated whether exposure of Eps8 KO mice to environmental enrichment (EE) may promote the morphological recovery of dendritic spine dysgenesis and density, thus opening the possibility that behavioural protocols may rescue synaptic defects with a genetic basis, possibly impinging on the same molecular pathways.

  • Results & Discussion

    Exposure to Environmental Enrichment rescues the spine defects of Eps8 KO mice

    To examine whether environmental stimulation reduces spine abnormalities in Eps8 deficient mice, Eps8 KO mice and their WT littermates were exposed to EE for 8 weeks. The treatment was started after weaning at P21 (postnatal day 21) because EE exposure during postnatal development (early EE) is known to have greater benefits than in adult mice. 8 to 10 WT and Eps8 KO- mice were housed together in a large cage having several different brightly coloured mouse toys and a running wheel (see methods for details). Control littermates were housed, inside the same room, in standard cages with only bedding and free access to water and food pellets (SH). At the end of the period, the impact of EE exposure on dendritic spine density and morphology was evaluated by Golgi-Cox staining as previously described. Quantification showed that, in WT mice, EE training induces a significant increase in the density of dendritic spines (Fig. 1A, B) and a decrease of spine length (Fig. 1A, C) in CA1 pyramidal neurons with respect to control SH-WT mice indicating that EE exposure affects spine number and morphogenesis. Of note a reduction of spine length may reflect a decreased number of immature spines, which display longer neck with respect to mature spines. In Eps8 KO mice EE training caused a significant reduction of spine density and length of CA1 pyramidal neurons with respect to control SH-Eps8 KO mice (Fig. 1A–C) thus leading to a partial recovery of the spine defects (i.e. abnormal number and shape) that characterized the Eps8 KO mice. These results show that EE exposure in Eps 8KO mice exerts a substantial rescue of the spine abnormalities suggesting that the synaptic defects due to Eps8 deficiency can be rescued by other factors possibly impinging on spare molecular mechanisms.

    To gain insights into the molecular mechanisms at the basis of the rescue of spine abnormalities in EE-exposed Eps8 KO mice, we assessed spine morphogenesis and plasticity in Eps8 KO cultures exposed to increased extracellular Mg2+ concentration ([Mg2+]e), which has been demonstrated to enhance synaptic plasticity in WT neurons by impacting calcium and NMDAR signalling. Results showed that Eps8 KO neurons grown in increased [Mg2+]e (up to 1.2 mM from 3 DIV old) display a recovered spine morphogenesis (Fig. 1D), as shown by the increased spine head size (Fig. 1E) and the higher number of mature mushroom-type spines (Fig. 1F), accompanied by a concomitant reduction of immature filopodia-like spines (Fig. 1G). To assess whether the morphological rescue is accompanied by a functional recovery, Eps8 KO neurons, exposed or not to increased extracellular concentration of magnesium, were subjected to chemically-induced NMDA-dependent LTP as described. Morphological analysis of the synapse potentiation was then assessed by evaluating the size of PSD-95 positive puncta as previously described. The results showed that elevation of extracellular Mg2+ concentration recovers Eps8 KO neuronal ability to undergo potentiation (Fig. 1H, I) indicating that increasing magnesium concentration ameliorates both spine maturation and plasticity.

  • Conclusions

    Eps8 KO mice and neurons display increased density of dendritic spines, which are morphologically immature, and defective synaptic signalling and plasticity. Here, we show that exposure of Eps8 deficient cultures or Eps8 KO mice to early environmental treatments (exposure to Mg or EE, respectively) corrects abnormal spine density and morphology and restores structural synaptic plasticity. These data suggest that the positive effects showed by early interventions in neuro-developmental diseases, such as the applied behavioural analysis, may rely on spared molecular pathways of synaptic plasticity.

  • Limitations

    While this study demonstrates that Eps8 KO mice, which were subjected to early EE, display a recovered synaptic structure in the hippocampal CA1 region as assessed by the quantification of spine number and shape (major core defects of this mouse model), testing cognitive abilities of EE treated-Eps8 mutant mice would complement this analysis. Furthermore, in this study, we tested only one protocol of EE that was administered immediately after weaning and comprises motor, sensor and social enrichment. Investigation of different modality of EE exposure in larger sample sizes would allow to discriminate among the best environmental manipulation in terms of specific recovery (structure vs function) in the future.

  • Methods

    Cell cultures

    Primary cultures of mouse hippocampal neurons were established from E18 foetal, Eps8 KO or wild type (wt) littermates C57BL/6 mice as described by. Briefly, hippocampi were dissociated by treatment with trypsin (0.125% for 15 min at 37°C), followed by trituration with a polished Pasteur pipette. The dissociated cells were plated onto glass coverslips coated with poly-L-lysine at density of 400 cells/mm2. The cells were maintained in Neurobasal (Invitrogen, San Diego, CA) with B27 supplement and antibiotics, 2 mM glutamine and 12.5 mM glutamate (neuronal medium).

    Chemical Long Term Potentiation (cLTP)

    Neuronal cultures were subjected to a chemical LTP protocol consisting of an application of high doses of glycine for 3 min. In particular, for the LTP induction cultured neurons were incubated in a KRH solution devoid of Mg2+ and containing (in mM: 0.1 mM Glycine, 125 NaCl, 5 KCl, 1.2 KH2PO4, 2 CaCl2, 6 glucose, and 25 HEPES-NaOH, TTX 0.001, Strychnine 0.001 and bicuculline methiodide 0.02, pH 7.4) for 3 min followed by a wash and recovery in neuronal medium for at least 60 min. After 60 min cells were immediately fixed and stained for PSD-95, v-Glut1, and tubulin.

    Transfection and Immunocytochemical staining

    Mouse hippocampal neurons were transfected with pSUPER-DsRed plasmid (obtained from pSUPER-GFP, Oligoengine, Seattle, WA, USA) at DIV13 using Lipofectamine 2000 (Invitrogen). Neuronal cultures were fixed with 4% paraformaldehyde + 4% sucrose at DIV15 as described. The following antibodies were used: guinea pig anti-vGLUT1 (1:1000; Synaptic Systems), mouse anti-PSD95 (1:400; UC Davis/NIH NeuroMab Facility, CA), rabbit anti-tubulin (1:80; Sigma-Aldrich, Milan, Italy). Secondary antibodies were conjugated with Alexa-488, Alexa-555, or Alexa-633 fluorophores (Invitrogen). Images were acquired using a Zeiss LSM 510 META confocal microscope producing image stacks. Pixel size was 110 × 110 nm, and acquisition parameters (i.e., laser power, gain and offset) were kept constant among different experimental settings. Furthermore, to overcome subjective bias during result analysis, we used an automated software-based methods of analysis, ImageJ software (NIH, Bethesda, MD, USA) keeping the parameters of the analyses constant among the different groups. In particular, dendritic spines were classified according the following parameters: mushroom (length ≤ 1.2 μm, width ≥0.5 μm); filopodia (length ˃1.2 μm, width ˂0.5 μm), in line with. Colocalization of 2 or 3 selected markers was measured using the boolean function ‘AND’ for the selected channels. The resulting image was binarized and used as a colocalization mask to be subtracted to single channels. The number of the puncta resulting from colocalization mask subtraction was measured for each marker. A colocalization ratio was set as colocalizing puncta/total puncta number.

    Exposure to environmental enrichment

    Environmental Enrichment has been performed as described with slight modifications. 8–10 WT and Eps8 KO mice were housed together for 8 weeks starting after weaning at P21 in a large cage (60 × 60 × 40 cm) with water and food ad libitum and several different brightly coloured mouse toys and a running wheel. To stimulate active exploration of a novel environment, new toys were swapped for existing once every week. The activity of the individual mice (exploring the objects and /or running on a wheel) was monitored daily. Control littermate mice were housed in the same room in standard cages with only bedding and access to water and food pellets (SH).

    Golgi staining and quantification of dendritic spines

    Mice were deeply anesthetized with chloral hydrate (4%; 1 ml/100 g body weight, i.p.) and subjected to intra-cardiac perfusion with 0.9% saline solution. The brains were removed stained by modified Golgi-Cox method as described in with slight modifications. Coronal sections of 100 mm thickness from the dorsal hippocampus were obtained using a vibratome (VT1000S, Leica, Wetzlar, Germany). These sections were collected free floating in 6% sucrose solution and processed with ammonium hydroxide for 15 min, followed by 15 min in Kodak Film Fixer, and finally were rinsed with distilled water, placed on coverslips, dehydrated and mounted with a xylene-based medium. Spine density and length was measured on the secondary branches of apical dendrites of pyramidal neurons located in the CA1 subfield of the dorsal hippocampus. At least 30 neurons per animal were evaluated.

    Statistical analysis

    Statistical analysis was performed using Prism6 (GraphPad), data are presented as mean±SEM from the indicated number of experiments. After testing whether data were normally distributed or not, the appropriate statistical test, followed by specific multiple comparison post hoc tests, has been used as indicated in figure legends. Kolmogorov–Smirnov test was used to determine significance in cumulative distributions of mEPSC amplitudes. Differences were considered to be significant if p<0.05 and are indicated by one asterisk; those at p<0.01 are indicated by double asterisks; those at p<0.001 are indicated by triple asterisks, those at p<0.0001 are indicated by four asterisks.

  • Funding statement

    We thank Monzino Foundation (Milano, Italy) for its generous gift of the Zeiss LSM800 confocal microscope to the section of Milan of the CNR Institute of Neuroscience. RM was supported by Fondazione Umberto Veronesi. EM was supported by Fondazione Vodafone Italia, Progetto Bandiera Interomics 2015-2017, Cariplo Rif. 2017-0622 and TERNA. MM was supported by Ministero della Salute GR-2011-02347377, Cariplo 2015-0594, Project “AMANDA” CUP_B42F16000440005 from Regione Lombardia and CNR Research Project on Aging, Fondazione Veronesi e Fondazione Vodafone Italia.

  • Ethics statement

    Procedures involving animals handling and care were conformed to protocols approved by the University of Milan in compliance with national (4D.L. N.116, G.U., suppl. 40, 18-2-1992) and international law and policies (EEC Council directive 2010/63/EU, OJ L 276/33, 22-09-2010; National Institutes of Health Guide for the Care and Use of Laboratory Animals, US National Research Council, 2011). All efforts were made to minimize the number of mice used and their suffering.

  • References
  • 1
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    Matters13.5/20

    Exposure to Environmental Factors rescues spine defects of Eps8 KO mice

    Affiliation listing not available.
    Abstractlink

    Several neuropsychiatric diseases, including autism spectrum disorders (ASD) and intellectual disability (ID) are characterized by synaptic dysfunctions, such as, altered number and shape of dendritic spines, and defective synaptic signalling and plasticity. The actin regulatory protein, Eps8, plays a key role in spine morphogenesis and plasticity since neurons lacking Eps8 show defective spine morphology and density and fail to undergo long term potentiation. Consistently, Eps8 KO mice display increased density of immature spines in CA1 hippocampal region and cognitive defects. Also, lower levels of Eps8 have been detected in the brain of a cohort of ASD patients. Preclinical studies showed that environmental factors, such as mice exposure to environmental enrichment (EE) or neuron exposure to increased extracellular Mg2+, may have a “plasticizing action” resulting in improved cognitive functions and higher cellular plasticity. Here, we tested whether early EE or high Mg2+ exposure may rescue the structural synaptic defects of Eps8 KO mice and neurons. Results of this study indicate that the early EE treatment in mice and elevated extracellular Mg2+ concentration in neurons restore normal dendritic morphology and synaptic plasticity in Eps8 KO mice and neurons. These data suggest that early “plasticizing” interventions can be beneficial on synaptic defects and their efficacy in the treatment of neuro-developmental diseases is worth to be investigated.

    Figurelink

    Figure 1.

    (A) Representative images of apical dendritic shaft of CA1 pyramidal neurons from WT and Eps8 KO mice exposed (EE) or not (SH) to the environmental enrichment protocol and stained by Golgi-Cox method. Scale bar, 10 mm.

    (B) Quantification of dendritic spine density and length (C) on the secondary branches of apical dendrites of CA1 pyramidal neurons. Number of spines/mm of parent dendrite: WT-SH=0.975±0.021; WT-EE=1.214±0.022; KO-SH=1.323±0.044; KO-EE=1.089±0.031. Spine length (in mm): WT-SH=1.299±0.015; WT-EE=1.146±0.009; KO-SH=1.405±0.014; KO-EE=1.108±0.013. Data are shown as mean±SEM. WT-SH: 102 cells, 6 mice; WT-EE: 160 cells, 8 mice; Eps8 KO-SH: 64 cells, 4 mice; Eps8 KO-EE: 60 cells, 4 mice. Mann-Whitney test **p<0,01; ***p<0,001. In addition a two-way ANOVA was conducted examining the effect of genotype and EE exposure on dendritic spine density and length. Results show that there is a statistically significant interaction between the effect of genotype and treatment on spine density ( F(1, 380) = 63,05; p<0,0001) and for the effect of genotype and treatment on spine length (F(1, 1016) 14,58, p=0,0001).

    (D) Representative confocal images of 14DIV WT and Eps8 KO primary hippocampal neurons, grown (+Mg) or not (CTR) in augmented extracellular Mg concentration, transfected with pSUPER-DsRed plasmid (red) and stained for PSD95 (green). Scale bar, 10 mm.

    (E) Quantification of spine width (in μm), percentage of immature-thin (F) and mature-mushroom (G) spines. Data are shown as mean±SEM. Kruskal-Wallis test, ****p<0.001; **p<0.1. Number of analyzed spines: WT-CTR=670, WT+Mg=664, KO-CTR=439, KO+Mg=524, 3 independent experiments.

    (H) Representative images of 14DIV Eps8 KO cultures treated or not with Mg2+ and subjected to chemical LTP protocol as indicated and stained for Tubulin (green), vGlut1 (blue) and PSD95 (red). Scale bar, 10 mm.

    (I) Quantification of PSD95 puncta size in different experimental conditions showing that Eps8 KO cultures grown in augmented extracellular Mg2+ concentration (up to 1.2 mM) are able to undergo LTP. Data are shown as normalized mean value±SEM. Mann-Whitney test. ***p<0.001. Number of fields examined=30; 3 independent experiments.

    Introductionlink

    Several neuropsychiatric diseases, including autism spectrum disorders (ASD) and intellectual disability (ID) are characterized by synaptic pathology, consisting in abnormal density and morphology of dendritic spines, synapse loss and aberrant synaptic signalling and plasticity[1][2]. For these reasons they are collectively called synaptopathies[3]. Of relevance, the dendritic spine dysgenesis found in individuals with autism related disorders[4] is consistently replicated in experimental mouse models[5].

    Dendritic spines are small actin-rich protrusions from neuronal dendrites that form the postsynaptic compartment of most excitatory synapses and are essential sites for learning and memory[6]. A tight regulation of the actin cytoskeleton is essential to the formation, maturation, and plasticity of dendritic spines[7][5]. Among the molecular players involved in this process, Eps8, a multi-functional actin-binding protein that regulates actin remodelling through Rac modulation and actin capping and bundling activities[8][9], has been found to play a key role in spine morphogenesis and plasticity. Eps8 is recruited to the spine head during LTP and inhibition of its capping activity impairs spine enlargement and plasticity[10]. Cultured neurons lacking Eps8 show an abnormal increased density of spines which display an immature filopodia-like shape and are unable to undergo morphological and functional synaptic potentiation upon chemical-LTP protocol. Also altered NMDA receptors composition has been found in Eps8 KO neurons[11]. Consistently, Eps8 knock out mice (Eps8 KO) display increased density of immature spines in CA1 hippocampal region and are impaired in cognitive functions. Of note reduced Eps8 levels have been detected in the brain of post-mortem ASD patients[10].

    Environmental enrichment, which provides enhanced sensory, cognitive and physical activity in rearing conditions[12][13] has been shown to improve cognitive functions, cellular plasticity and associated molecular processes in mouse models of brain diseases[14][15]. Also, it has been proposed as an effective treatment for ASD[16][17]. Indeed, the behavioural therapy – such as applied behavioural analysis (ABA) - is usually the first-line treatment of children affected by ASD that experience difficulty in developing social, speech, and behavioural skills[18][19][20], while the pharmacological therapies help patients function in their daily activities[21]. In particular, risperidone and aripiprazole, which remain the pillars of ASD treatment, are FDA-approved drugs only for managing the irritability associated with the disorder[21].

    Objectivelink

    Based on these premises, we investigated whether exposure of Eps8 KO mice to environmental enrichment (EE) may promote the morphological recovery of dendritic spine dysgenesis and density, thus opening the possibility that behavioural protocols may rescue synaptic defects with a genetic basis, possibly impinging on the same molecular pathways.

    Results & Discussionlink

    Exposure to Environmental Enrichment rescues the spine defects of Eps8 KO mice

    To examine whether environmental stimulation reduces spine abnormalities in Eps8 deficient mice, Eps8 KO mice and their WT littermates were exposed to EE for 8 weeks. The treatment was started after weaning at P21 (postnatal day 21) because EE exposure during postnatal development (early EE) is known to have greater benefits than in adult mice[22][23]. 8 to 10 WT and Eps8 KO- mice were housed together in a large cage having several different brightly coloured mouse toys and a running wheel (see methods for details). Control littermates were housed, inside the same room, in standard cages with only bedding and free access to water and food pellets (SH). At the end of the period, the impact of EE exposure on dendritic spine density and morphology was evaluated by Golgi-Cox staining as previously described[10]. Quantification showed that, in WT mice, EE training induces a significant increase in the density of dendritic spines (Fig. 1A, B) and a decrease of spine length (Fig. 1A, C) in CA1 pyramidal neurons with respect to control SH-WT mice indicating that EE exposure affects spine number and morphogenesis. Of note a reduction of spine length may reflect a decreased number of immature spines, which display longer neck with respect to mature spines[24]. In Eps8 KO mice EE training caused a significant reduction of spine density and length of CA1 pyramidal neurons with respect to control SH-Eps8 KO mice (Fig. 1A–C) thus leading to a partial recovery of the spine defects (i.e. abnormal number and shape) that characterized the Eps8 KO mice. These results show that EE exposure in Eps 8KO mice exerts a substantial rescue of the spine abnormalities suggesting that the synaptic defects due to Eps8 deficiency can be rescued by other factors possibly impinging on spare molecular mechanisms.

    To gain insights into the molecular mechanisms at the basis of the rescue of spine abnormalities in EE-exposed Eps8 KO mice, we assessed spine morphogenesis and plasticity in Eps8 KO cultures exposed to increased extracellular Mg2+ concentration ([Mg2+]e), which has been demonstrated to enhance synaptic plasticity in WT neurons by impacting calcium and NMDAR signalling[25]. Results showed that Eps8 KO neurons grown in increased [Mg2+]e (up to 1.2 mM from 3 DIV old) display a recovered spine morphogenesis (Fig. 1D), as shown by the increased spine head size (Fig. 1E) and the higher number of mature mushroom-type spines (Fig. 1F), accompanied by a concomitant reduction of immature filopodia-like spines (Fig. 1G). To assess whether the morphological rescue is accompanied by a functional recovery, Eps8 KO neurons, exposed or not to increased extracellular concentration of magnesium, were subjected to chemically-induced NMDA-dependent LTP as described[26]. Morphological analysis of the synapse potentiation was then assessed by evaluating the size of PSD-95 positive puncta as previously described[26][27][10]. The results showed that elevation of extracellular Mg2+ concentration recovers Eps8 KO neuronal ability to undergo potentiation (Fig. 1H, I) indicating that increasing magnesium concentration ameliorates both spine maturation and plasticity.

    Conclusionslink

    Eps8 KO mice and neurons display increased density of dendritic spines, which are morphologically immature, and defective synaptic signalling and plasticity. Here, we show that exposure of Eps8 deficient cultures or Eps8 KO mice to early environmental treatments (exposure to Mg or EE, respectively) corrects abnormal spine density and morphology and restores structural synaptic plasticity. These data suggest that the positive effects showed by early interventions in neuro-developmental diseases, such as the applied behavioural analysis, may rely on spared molecular pathways of synaptic plasticity.

    Limitationslink

    While this study demonstrates that Eps8 KO mice, which were subjected to early EE, display a recovered synaptic structure in the hippocampal CA1 region as assessed by the quantification of spine number and shape (major core defects of this mouse model), testing cognitive abilities of EE treated-Eps8 mutant mice would complement this analysis. Furthermore, in this study, we tested only one protocol of EE that was administered immediately after weaning and comprises motor, sensor and social enrichment. Investigation of different modality of EE exposure in larger sample sizes would allow to discriminate among the best environmental manipulation in terms of specific recovery (structure vs function) in the future.

    Methodslink

    Cell cultures

    Primary cultures of mouse hippocampal neurons were established from E18 foetal, Eps8 KO or wild type (wt) littermates C57BL/6 mice as described by[10]. Briefly, hippocampi were dissociated by treatment with trypsin (0.125% for 15 min at 37°C), followed by trituration with a polished Pasteur pipette. The dissociated cells were plated onto glass coverslips coated with poly-L-lysine at density of 400 cells/mm2. The cells were maintained in Neurobasal (Invitrogen, San Diego, CA) with B27 supplement and antibiotics, 2 mM glutamine and 12.5 mM glutamate (neuronal medium).

    Chemical Long Term Potentiation (cLTP)

    Neuronal cultures were subjected to a chemical LTP protocol[26] consisting of an application of high doses of glycine for 3 min. In particular, for the LTP induction cultured neurons were incubated in a KRH solution devoid of Mg2+ and containing (in mM: 0.1 mM Glycine, 125 NaCl, 5 KCl, 1.2 KH2PO4, 2 CaCl2, 6 glucose, and 25 HEPES-NaOH, TTX 0.001, Strychnine 0.001 and bicuculline methiodide 0.02, pH 7.4) for 3 min followed by a wash and recovery in neuronal medium for at least 60 min. After 60 min cells were immediately fixed and stained for PSD-95, v-Glut1, and tubulin.

    Transfection and Immunocytochemical staining

    Mouse hippocampal neurons were transfected with pSUPER-DsRed plasmid (obtained from pSUPER-GFP, Oligoengine, Seattle, WA, USA) at DIV13 using Lipofectamine 2000 (Invitrogen). Neuronal cultures were fixed with 4% paraformaldehyde + 4% sucrose at DIV15 as described[28]. The following antibodies were used: guinea pig anti-vGLUT1 (1:1000; Synaptic Systems), mouse anti-PSD95 (1:400; UC Davis/NIH NeuroMab Facility, CA), rabbit anti-tubulin (1:80; Sigma-Aldrich, Milan, Italy). Secondary antibodies were conjugated with Alexa-488, Alexa-555, or Alexa-633 fluorophores (Invitrogen). Images were acquired using a Zeiss LSM 510 META confocal microscope producing image stacks. Pixel size was 110 × 110 nm, and acquisition parameters (i.e., laser power, gain and offset) were kept constant among different experimental settings. Furthermore, to overcome subjective bias during result analysis, we used an automated software-based methods of analysis, ImageJ software (NIH, Bethesda, MD, USA) keeping the parameters of the analyses constant among the different groups. In particular, dendritic spines were classified according the following parameters: mushroom (length ≤ 1.2 μm, width ≥0.5 μm); filopodia (length ˃1.2 μm, width ˂0.5 μm), in line with[29]. Colocalization of 2 or 3 selected markers was measured using the boolean function ‘AND’ for the selected channels. The resulting image was binarized and used as a colocalization mask to be subtracted to single channels. The number of the puncta resulting from colocalization mask subtraction was measured for each marker. A colocalization ratio was set as colocalizing puncta/total puncta number.

    Exposure to environmental enrichment

    Environmental Enrichment has been performed as described with slight modifications[30]. 8–10 WT and Eps8 KO mice were housed together for 8 weeks starting after weaning at P21 in a large cage (60 × 60 × 40 cm) with water and food ad libitum and several different brightly coloured mouse toys and a running wheel. To stimulate active exploration of a novel environment, new toys were swapped for existing once every week. The activity of the individual mice (exploring the objects and /or running on a wheel) was monitored daily. Control littermate mice were housed in the same room in standard cages with only bedding and access to water and food pellets (SH).

    Golgi staining and quantification of dendritic spines

    Mice were deeply anesthetized with chloral hydrate (4%; 1 ml/100 g body weight, i.p.) and subjected to intra-cardiac perfusion with 0.9% saline solution. The brains were removed stained by modified Golgi-Cox method as described in[10][31] with slight modifications. Coronal sections of 100 mm thickness from the dorsal hippocampus were obtained using a vibratome (VT1000S, Leica, Wetzlar, Germany). These sections were collected free floating in 6% sucrose solution and processed with ammonium hydroxide for 15 min, followed by 15 min in Kodak Film Fixer, and finally were rinsed with distilled water, placed on coverslips, dehydrated and mounted with a xylene-based medium. Spine density and length was measured on the secondary branches of apical dendrites of pyramidal neurons located in the CA1 subfield of the dorsal hippocampus. At least 30 neurons per animal were evaluated.

    Statistical analysis

    Statistical analysis was performed using Prism6 (GraphPad), data are presented as mean±SEM from the indicated number of experiments. After testing whether data were normally distributed or not, the appropriate statistical test, followed by specific multiple comparison post hoc tests, has been used as indicated in figure legends. Kolmogorov–Smirnov test was used to determine significance in cumulative distributions of mEPSC amplitudes. Differences were considered to be significant if p<0.05 and are indicated by one asterisk; those at p<0.01 are indicated by double asterisks; those at p<0.001 are indicated by triple asterisks, those at p<0.0001 are indicated by four asterisks.

    Funding Statementlink

    We thank Monzino Foundation (Milano, Italy) for its generous gift of the Zeiss LSM800 confocal microscope to the section of Milan of the CNR Institute of Neuroscience. RM was supported by Fondazione Umberto Veronesi. EM was supported by Fondazione Vodafone Italia, Progetto Bandiera Interomics 2015-2017, Cariplo Rif. 2017-0622 and TERNA. MM was supported by Ministero della Salute GR-2011-02347377, Cariplo 2015-0594, Project “AMANDA” CUP_B42F16000440005 from Regione Lombardia and CNR Research Project on Aging, Fondazione Veronesi e Fondazione Vodafone Italia.

    Conflict of interestlink

    The authors declare no conflicts of interest.

    Ethics Statementlink

    Procedures involving animals handling and care were conformed to protocols approved by the University of Milan in compliance with national (4D.L. N.116, G.U., suppl. 40, 18-2-1992) and international law and policies (EEC Council directive 2010/63/EU, OJ L 276/33, 22-09-2010; National Institutes of Health Guide for the Care and Use of Laboratory Animals, US National Research Council, 2011). All efforts were made to minimize the number of mice used and their suffering.

    No fraudulence is committed in performing these experiments or during processing of the data. We understand that in the case of fraudulence, the study can be retracted by ScienceMatters.

    Referenceslink
    1. Myrrhe van Spronsen, Casper C. Hoogenraad
      Synapse Pathology in Psychiatric and Neurologic Disease
      Current Neurology and Neuroscience Reports, 10/2010, pages 207-214 DOI: 10.1007/s11910-010-0104-8chrome_reader_mode
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