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Development of inhibitory synaptic inputs on layer 2/3 pyramidal neurons in the rat medial prefrontal cortex

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Abstract

Inhibitory control of pyramidal neurons plays a major role in governing the excitability in the brain. While spatial mapping of inhibitory inputs onto pyramidal neurons would provide important structural data on neuronal signaling, studying their distribution at the single cell level is difficult due to the lack of easily identifiable anatomical proxies. Here, we describe an approach where in utero electroporation of a plasmid encoding for fluorescently tagged gephyrin into the precursors of pyramidal cells along with ionotophoretic injection of Lucifer Yellow can reliably and specifically detect GABAergic synapses on the dendritic arbour of single pyramidal neurons. Using this technique and focusing on the basal dendritic arbour of layer 2/3 pyramidal cells of the medial prefrontal cortex, we demonstrate an intense development of GABAergic inputs onto these cells between postnatal days 10 and 20. While the spatial distribution of gephyrin clusters was not affected by the distance from the cell body at postnatal day 10, we found that distal dendritic segments appeared to have a higher gephyrin density at later developmental stages. We also show a transient increase around postnatal day 20 in the percentage of spines that are carrying a gephyrin cluster, indicative of innervation by a GABAergic terminal. Since the precise spatial arrangement of synaptic inputs is an important determinant of neuronal responses, we believe that the method described in this work may allow a better understanding of how inhibition settles together with excitation, and serve as basics for further modelling studies focusing on the geometry of dendritic inhibition during development.

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References

  • Beaulieu C, Kisvarday Z, Somogyi P, Cynader M, Cowey A (1992) Quantitative distribution of GABA-immunopositive and -immunonegative neurons and synapses in the monkey striate cortex (area 17). Cereb Cortex 2:295–309

    Article  CAS  PubMed  Google Scholar 

  • Briner A, Nikonenko I, De Roo M, Dayer A, Muller D, Vutskits L (2011) Developmental stage-dependent persistent impact of propofol anesthesia on dendritic spines in the rat medial prefrontal cortex. Anesthesiology 115:282–293

    Article  CAS  PubMed  Google Scholar 

  • Brockmann MD, Pöschel B, Cichon N, Hanganu-Opatz IL (2011) Coupled oscillations mediate directed interactions between prefrontal cortex and hippocampus of the neonatal rat. Neuron 71:332–347

    Article  CAS  PubMed  Google Scholar 

  • Chen JL, Villa KL, Cha JW, So PT, Kubota Y, Nedivi E (2012) Clustered dynamics of inhibitory synapses and dendritic spines in the adult neocortex. Neuron 74:361–373

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chiu CQ, Lur G, Morse TM, Carnevale NT, Ellis-Davies GC, Higley MJ (2013) Compartmentalization of GABAergic inhibition by dendritic spines. Science 340:759–762

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dammerman RS, Flint AC, Noctor S, Kriegstein AR (2000) An excitatory GABAergic plexus in developing neocortical layer 1. J Neurophysiol 84:428–434

    Article  CAS  PubMed  Google Scholar 

  • De Felipe J, Marco P, Fairen A, Jones EG (1997) Inhibitory synaptogenesis in mouse somatosensory cortex. Cereb Cortex 7:619–634

    Article  PubMed  Google Scholar 

  • Fiumelli H, Briner A, Puskarjov M, Blaesse P, Belem BJ, Dayer AG, Kaila K, Martin JL, Vutskits L (2013) An ion transport-independent role for the cation-chloride cotransporter KCC2 in dendritic spinogenesis in vivo. Cereb Cortex 23:378–388

    Article  PubMed  Google Scholar 

  • Flores CE, Nikonenko I, Mendez P, Fritschy JM, Tyagarajan SK, Muller D (2015) Activity-dependent inhibitory synapse remodeling through gephyrin phosphorylation. Proc Natl Acad Sci USA 112:E65–E72

    Article  CAS  PubMed  Google Scholar 

  • Gidon A, Segev I (2012) Principles governing the operation of synaptic inhibition in dendrites. Neuron 75:330–341

    Article  CAS  PubMed  Google Scholar 

  • Gonzalez-Burgos G, Hashimoto T, Lewis DA (2010) Alterations of cortical GABA neurons and network oscillations in schizophrenia. Curr Psychiatry Rep 12:335–344

    Article  PubMed  PubMed Central  Google Scholar 

  • Gulyás AI, Megías M, Emri Z, Freund TF (1999) Total number and ratio of excitatory and inhibitory synapses converging onto single interneurons of different types in the CA1 area of the rat hippocampus. J Neurosci 19:10082–10097

    PubMed  Google Scholar 

  • Hao J, Wang XD, Dan Y, Poo MM, Zhang XH (2009) An arithmetic rule for spatial summation of excitatory and inhibitory inputs in pyramidal neurons. Proc Natl Acad Sci USA 106:21906–21911

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jasinska M, Siucinska E, Cybulska-Klosowicz A, Pyza E, Furness DN, Kossut M, Glazewski S (2010) Rapid, learning-induced inhibitory synaptogenesis in murine barrel field. J Neurosci 30:1176–1184

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jones EG, Powell TP (1969) Morphological variations in the dendritic spines of the neocortex. J Cell Sci 5:509–529

    CAS  PubMed  Google Scholar 

  • Kaila K, Price TJ, Payne JA, Puskarjov M, Voipio J (2014) Cation-chloride cotransporters in neuronal development, plasticity and disease. Nat Rev Neurosci 15:637–654

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Klausberger T, Somogyi P (2008) Neuronal diversity and temporal dynamics: the unity of hippocampal circuit operations. Science 321:53–57

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kubota Y, Hatada S, Kondo S, Karube F, Kawaguchi Y (2007) Neocortical inhibitory terminals innervate dendritic spines targeted by thalamocortical afferents. J Neurosci 27:1139–1150

    Article  CAS  PubMed  Google Scholar 

  • Kubota Y, Karube F, Nomura M, Kawaguchi Y (2016) The diversity of cortical inhibitory synapses. Front Neural Circuits 10:27

    Article  PubMed  PubMed Central  Google Scholar 

  • Lardi-Studler B, Smolinsky B, Petitjean CM, Koenig F, Sidler C, Meier JC, Fritschy JM, Schwarz G (2007) Vertebrate-specific sequences in the gephyrin E-domain regulate cytosolic aggregation and postsynaptic clustering. J Cell Sci 120:1371–1382

    Article  CAS  PubMed  Google Scholar 

  • Maas C, Tagnaouti N, Loebrich S, Behrend B, Lappe-Siefke C, Kneussel M (2006) Neuronal cotransport of glycine receptor and the scaffold protein gephyrin. J Cell Biol 172:441–451

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • McGlashan TH, Hoffman RE (2000) Schizophrenia as a disorder of developmentally reduced synaptic connectivity. Arch Gen Psychiatry 57:637–648

    Article  CAS  PubMed  Google Scholar 

  • Megías M, Emri Z, Freund TF, Gulyás AI (2001) Total number and distribution of inhibitory and excitatory synapses on hippocampal CA1 pyramidal cells. Neuroscience 102:527–540

    Article  PubMed  Google Scholar 

  • Micheva KD, Beaulieu C (1996) Quantitative aspects of synaptogenesis in the rat barrel field cortex with special reference to GABA circuitry. J Comp Neurol 373:340–354

    Article  CAS  PubMed  Google Scholar 

  • Müller C, Beck H, Coulter D, Remy S (2012) Inhibitory control of linear and supralinear dendritic excitation in CA1 pyramidal neurons. Neuron 75:851–864

    Article  PubMed  Google Scholar 

  • Paxinos G, Watson C (1998) The rat brain in stereotaxic coordinates. Academic Press, San Diego

    Google Scholar 

  • Penzes P, Cahill ME, Jones KA, VanLeeuwen JE, Woolfrey KM (2011) Dendritic spine pathology in neuropsychiatric disorders. Nat Neurosci 14:285–293

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Puskarjov M, Fiumelli H, Briner A, Bodogan T, Demeter K, Lacoh CM, Mavrovic M, Blaesse P, Kaila K, Vutskits L (2017) K–Cl cotransporter 2-mediated Cl− extrusion determines developmental stage-dependent impact of propofol anesthesia on dendritic spines. Anesthesiology 126:855–867

    Article  CAS  PubMed  Google Scholar 

  • Rieubland S, Roth A, Häusser M (2012) Dendritic ventriloquism: inhibitory synapses throw their voices. Neuron 75:190–193

    Article  CAS  PubMed  Google Scholar 

  • Saito T, Nakatsuji N (2001) Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev Biol 240:237–246

    Article  CAS  PubMed  Google Scholar 

  • Shepherd G, Grillner S (2010) Handbook of brain microcircuits. Oxford University Press, New York

    Book  Google Scholar 

  • Sholl DA (1953) Dendritic organization in the neurons of the visual andmotor cortices of the cat. J Anat 87:387–406

    CAS  PubMed  PubMed Central  Google Scholar 

  • Specht CG, Izeddin I, Rodriguez PC, El Beheiry M, Rostaing P, Darzacq X, Dahan M, Triller A (2013) Quantitative nanoscopy of inhibitory synapses: counting gephyrin molecules and receptor binding sites. Neuron 79:308–321

    Article  CAS  PubMed  Google Scholar 

  • Spruston N (2008) Pyramidal neurons: dendritic structure and synaptic integration. Nat Rev Neurosci 9:206–221

    Article  CAS  PubMed  Google Scholar 

  • Studer R, von Boehmer L, Haenggi T, Schweizer C, Benke D, Rudolph U, Fritschy JM (2006) Alteration of GABAergic synapses and gephyrin clusters in the thalamic reticular nucleus of GABAA receptor alpha3 subunit-null mice. Eur J Neurosci 24:1307–1315

    Article  PubMed  Google Scholar 

  • Takesian AE, Hensch TK (2013) Balancing plasticity/stability across brain development. Prog Brain Res 207:3–34

    Article  PubMed  Google Scholar 

  • Tsujimoto S (2008) The prefrontal cortex: functional neural development during early childhood. Neuroscientist 14:345–358

    Article  PubMed  Google Scholar 

  • Tyagarajan SK, Fritschy JM (2014) Gephyrin: a master regulator of neuronal function. Nat Rev Neurosci 15:141–156

    Article  CAS  PubMed  Google Scholar 

  • Tyagarajan SK, Ghosh H, Harvey K, Fritschy JM (2011a) Collybistin splice variants differentially interact with gephyrin and Cdc42 to regulate gephyrin clustering at GABAergic synapses. J Cell Sci 124:2786–2796

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tyagarajan SK, Ghosh H, Yevenes GE, Nikonenko I, Ebeling C, Schwerdel C, Sidler C, Zeilhofer HU, Gerrits B, Muller D, Fritschy JM (2011b) Regulation of GABAergic synapse formation and plasticity by GSK3beta-dependent phosphorylation of gephyrin. Proc Natl Acad Sci USA 108:379–384

    Article  CAS  PubMed  Google Scholar 

  • Uylings HB, Groenewegen HJ, Kolb B (2003) Do rats have a prefrontal cortex? Behav Brain Res 146:3–17

    Article  PubMed  Google Scholar 

  • van Versendaal D, Rajendran R, Saiepour MH, Klooster J, Smit-Rigter L, Sommeijer JP, De Zeeuw CI, Hofer SB, Heimel JA, Levelt CN (2012) Elimination of inhibitory synapses is a major component of adult ocular dominance plasticity. Neuron 74:374–383

    Article  PubMed  Google Scholar 

  • Villa KL, Berry KP, Subramanian J, Cha JW, Oh WC, Kwon HB, Kubota Y, So PT, Nedivi E (2016) Inhibitory synapses are repeatedly assembled and removed at persistent sites. Vivo Neuron 89:756–769

    Article  CAS  PubMed  Google Scholar 

  • Yang JM, Zhang J, Yu YQ, Duan S, Li XM (2014) Postnatal development of 2 microcircuits involving fast-spiking interneurons in the mouse prefrontal cortex. Cereb Cortex 24:98–109

    Article  PubMed  Google Scholar 

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Acknowledgements

We thank Michèle Brunet for excellent technical assistance, Kai Kaila, Jean-Marc Fritschy and Jozsef Kiss for instructive comments on the manuscript. This study was funded by the Swiss National Science Foundation, Berne, Switzerland, Grant 31003A-130625 (to LV).

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Author notes

  1. Mari A. Virtanen

    Present address: Department of Biosciences, University of Helsinki, Helsinki, Finland

Authors and Affiliations

  1. Department of Basic Neuroscience, University of Geneva Medical School, Geneva, Switzerland

    Mari A. Virtanen, Claudia Marvine Lacoh, Mathias de Roo & Laszlo Vutskits

  2. Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia

    Hubert Fiumelli

  3. Department of Psychiatry, University of Geneva Medical School, Geneva, Switzerland

    Markus Kosel

  4. Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland

    Shiva Tyagarajan

  5. Department of Anesthesiology, Pharmacology and Intensive Care, University Hospitals of Geneva, 4, rue Gabrielle-Perret-Gentil, 1211, Geneva 4, Switzerland

    Mathias de Roo & Laszlo Vutskits

Authors
  1. Mari A. Virtanen
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  2. Claudia Marvine Lacoh
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Contributions

MAV, MDR, MK, ST, JMF, and LV designed the experiments. MAV, MDR, CML, HF and LV performed and analyzed the experiments. MAV, MDR and LV wrote the manuscript with input from the co-authors.

Corresponding author

Correspondence to Laszlo Vutskits.

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429_2017_1602_MOESM1_ESM.tif

Supplementary material 1 Electronic Supplementary Figure 1. Gephyrin clusters were detected manually in image stacks, and judged to be inside the dendrite if the brightest pixel of the cluster colocalized with the Lucifer Yellow injection. A: Raw data showing a part of an image stack with Lucifer Yellow injection and gephyrin clusters imaged in separate channels. On the right side, the channel for gephyrin clusters is thresholded by offsetting the zero intensity level, to find the centre of each cluster. If the brightest part of the cluster colocalized with the Lucifer Yellow injection, the cluster was judged to be inside the dendrite (white arrows), otherwise the cluster was not included in the analysis (blue arrows). B: Maximum projection of the same image stack shows that colocalization cannot reliably be done in z-projected images. After projecting the image stack, some of the clusters previously judged to be outside the dendrite (blue arrows) will now appear to colocalize with the Lucifer Yellow injection. Scale bars: 1 μm (TIF 27829 KB)

Supplementary material 2 (PDF 32 KB)

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Virtanen, M.A., Lacoh, C.M., Fiumelli, H. et al. Development of inhibitory synaptic inputs on layer 2/3 pyramidal neurons in the rat medial prefrontal cortex. Brain Struct Funct 223, 1999–2012 (2018). https://doi.org/10.1007/s00429-017-1602-0

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