KCC2 Regulates Dendritic Spine Formation in a Brain-Region Specific and BDNF Dependent Manner

Cerebral Cortex, November 2018;28: 4049–4062 doi: 10.1093/cercor/bhy198 Advance Access Publication Date: 31 August 2018 Original Article ORIGINAL ARTICLE Patricia Nora Awad1,2, Clara Akofa Amegandjin1,2, Joanna Szczurkowska3, Josianne Nuñes Carriço2, Antônia Samia Fernandes do Nascimento2, Elie Baho1,2, Bidisha Chattopadhyaya1,2, Laura Cancedda3,4, Lionel Carmant1,2 and Graziella Di Cristo1,2 1 Department of Neurosciences, Université de Montréal, Montréal, Québec, Canada H3C 3J7, 2CHU SainteJustine Research Center, Montréal, Québec, Canada H3T 1C5, 3Neuroscience and Brain Technologies, Instituto Italiano di Tecnologia, Genova 16163, Italy and 4Telethon Dulbecco Institute, Italy Address correspondence to Graziella Di Cristo, Research Center, CHU Sainte-Justine/Université de Montréal, Neuroscience Department, 3175 Côte-SainteCatherine, Montréal, Québec, Canada H3T 1C5. Email: Abstract KCC2 is the major chloride extruder in neurons. The spatiotemporal regulation of KCC2 expression orchestrates the developmental shift towards inhibitory GABAergic drive and the formation of glutamatergic synapses. Whether KCC2’s role in synapse formation is similar in different brain regions is unknown. First, we found that KCC2 subcellular localization, but not overall KCC2 expression levels, differed between cortex and hippocampus during the first postnatal week. We performed site-specific in utero electroporation of KCC2 cDNA to target either hippocampal CA1 or somatosensory cortical pyramidal neurons. We found that a premature expression of KCC2 significantly decreased spine density in CA1 neurons, while it had the opposite effect in cortical neurons. These effects were cell autonomous, because single-cell biolistic overexpression of KCC2 in hippocampal and cortical organotypic cultures also induced a reduction and an increase of dendritic spine density, respectively. In addition, we found that the effects of its premature expression on spine density were dependent on BDNF levels. Finally, we showed that the effects of KCC2 on dendritic spine were dependent on its chloride transporter function in the hippocampus, contrary to what was observed in cortex. Altogether, these results demonstrate that KCC2 regulation of dendritic spine development, and its underlying mechanisms, are brain-region specific. Key words: brain-derived neurotrophic factor, dendritic spines, development, KCC2, synapse formation Introduction Proper function of neural circuits requires the orchestrated formation of trillions of synapses. During the last decades, much effort has been directed towards understanding how synapses form, a necessary step to better comprehend how the brain is built during normal development and how this process goes awry in diseases characterized by altered synapse function. The vast majority of studies have often focused on specific neuron cell types from specific brain regions. However, one important question is whether molecular mechanisms © The Author(s) 2018. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact KCC2 Regulates Dendritic Spine Formation in a BrainRegion Specific and BDNF Dependent Manner 4050 | Cerebral Cortex, 2018, Vol. 28, No. 11 Materials and Methods Animals Sprague–Dawley pups were obtained from Charles River Laboratories (St. Constant, Quebec, Canada) at postnatal day 1 (P1) or pregnant at gestational day 10 (E10). Pups were culled to 12 per dam, matched by gender, weighed and kept with their mother in a 12 h light/dark cycle with food and water ad libitum. Animal care and use conformed to institutional policies and guidelines (CIBPAR, Sainte-Justine Hospital Research Centre, Université de Montréal, Montreal, Quebec, Canada). Western Blot Whole lysate proteins were extracted from hippocampal and somatosensory cortex tissue in vivo or hippocampal and cortical organotypic cultures, at different ages in vivo or days in vitro, as indicated in the legends, following the previously described protocol (Ouardouz et al. 2010). To obtain membrane protein fractions, samples were homogenized in 5 vol of HB (300 mM sucrose/10 mM Tris–HCl, pH 7.5/1 mM EDTA/protease inhibitor mixture) and centrifuged at 1000 × g for 10 min at 4 °C. The pellet was washed in 0.5 vol HB and used as the nuclear fraction. The supernatants was centrifuged at 17 000 × g for 15 min at 4 °C, yielding the mitochondria fraction. The supernatant was further separated by ultracentrifugation at 100 000 × g for 1 h. The pellet and supernatant were used as the membrane and cytosol fractions, respectively. For organotypic slice cultures, at least 3–4 slices per animal were pooled in a sample, in order to have enough proteins from whole lysates. Each experimental group included 3–5 animals. Membranes were probed with the following primary antibodies: anti-KCC2 1:1000 (rabbit polyclonal IgG; Cat. no. 07-432, Millipore), 1:200 anti-BDNF (cat#N-20: sc-546, Santa-Cruz Biotechnology, Inc.), anti-glyceraldehyde-3-phosphate dehydrogenase 1:4000 (GAPDH, mouse monoclonal IgG; Cat. no. AM4300; Applied Biosystems) and anti-β dystroglycan 1:3000 (rabbit polyclonal IgG, Cat no. ab43125, Abcam). Specificity of BDNF antibody was verified using tissue from a BDNF−/− mouse and a wild-type littermate (data not shown). All samples were run simultaneously. Bands were quantified using Image J software. The intensity of KCC2 and BDNF bands were normalized over the intensity of the GAPDH band for whole lysates or of the β dystroglycan band for membrane fractions, in the same lane (internal loading control). Immunolabeling Experiments Brains were perfused with saline (0.9% NaCl) followed by 4% paraformaldehyde/phosphate buffer, pH 7.4, then cryoprotected in 30% sucrose/PBS, and frozen in Tissue Tek. Brains were sectioned (80 μm thick for in utero electroporation experiment, 40 μm for immunostaining of KCC2) using a cryostat (Leica). Slices were blocked in 10% NGS and 0.3% Triton for 2 h at room temperature, and incubated overnight at 4 °C in 5% NGS, 0.1% Triton and the following primary antibodies—NeuN, 1:400 (mouse monoclonal, Cat. no. MAB377 Millipore); KCC2, 1:200 (rabbit polyclonal, Cat. no. 07-432 Millipore); GFP, 1:500 (chicken polyclonal, Cat. no. Ab13970 Abcam). The following secondary antibodies were used: anti-mouse Alexa 633conjugated goat IgG and anti-rabbit Alexa 488-conjugated goat IgG (1:400; Molecular Probes, Invitrogen) or Alexa 488 goat anti-chicken IgY H&L (1:500, Cat. no. Ab150169 Abcam). NeuN staining was used to unequivocally identify the CA1 region in hippocampal slices. GFP immunostaining was used to enhance regulating synapse formation in a specific neuronal cell type (e.g., glutamatergi (...truncated)