Development of inhibitory synaptic inputs on layer 2/3 pyramidal neurons in the rat medial prefrontal cortex
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.
KeywordsDevelopment Cerebral cortex GABA Gephyrin Synaptogenesis
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).
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.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- Paxinos G, Watson C (1998) The rat brain in stereotaxic coordinates. Academic Press, San DiegoGoogle 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 126:855–867CrossRefPubMedGoogle 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 108:379–384CrossRefPubMedGoogle Scholar