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

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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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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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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Electronic supplementary material

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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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Keywords

  • Development
  • Cerebral cortex
  • GABA
  • Gephyrin
  • Synaptogenesis