Expression of Farnesylated EGFP in Primary Neocortex Culture Neurons Results in Impairs Dendritic Spike Development

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Resumo

Genetically encoded fluorescent proteins are widely used in biological research in general and in neurobiology in particular. When using these tools, it is important that the expression of the fluorescent protein does not disrupt the natural physiological processes in the cell. Addition of the farnesylation motif to fluorescent proteins leads to their anchoring in the plasma membrane, which is often used to visualize fine details of cell morphology, such as dendritic spines. In our work, we investigated the development of spines in primary cultured neocortical neurons by transfecting cells with farnesylated and unmodified EGFP by electroporation in suspension on the day of planting. It was found that neurons expressing farnesylated EGFP demonstrate pronounced disturbances in spine development, in particular, these cells were characterized by longer spines with more filopodia-like structures, which is typical for various pathological conditions. Therefore, when using farnesylated fluorescent proteins in experiments, it is necessary to take into account their possible negative impact on the development of various membrane structures of the cell, in particular neuronal spines.

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Sobre autores

G. Smirnova

Institute of Higher Nervous Activity and Neurophysiology, RAS

Email: malyshev@ihna.ru
Rússia, Moscow

O. Idzhilova

Institute of Higher Nervous Activity and Neurophysiology, RAS

Email: malyshev@ihna.ru
Rússia, Moscow

A. Abonakour

Institute of Higher Nervous Activity and Neurophysiology, RAS

Email: malyshev@ihna.ru
Rússia, Moscow

A. Malyshev

Institute of Higher Nervous Activity and Neurophysiology, RAS

Autor responsável pela correspondência
Email: malyshev@ihna.ru
Rússia, Moscow

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2. Fig. 1. Morphology of dendritic spines. a – Schematic representation of a spine with displayed parameters that were used to classify the spine into one of four morphological types: mushroom, stump, thin and filopodium. b – Confocal micrograph showing a fragment of a dendrite of a pyramidal neuron of the mouse neocortex transduced in vivo with an adeno-associated virus carrying farnesylated EGFP. Calibration bar is 2 μm.

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3. Fig. 2. Cultured neurons transfected with farnesylated EGFP exhibit pronounced developmental abnormalities compared to cells expressing unmodified EGFP. a, b – fragments of dendrites of neurons expressing fEGFP (a) and EGFP (c). Arrows with letters indicate examples of spines assigned to one or another morphological class: F – filopodium, G – mushroom-shaped, P – stump-shaped, T – thin. b, d – bodies of cells transfected with a plasmid with fEGFP (b) and EGFP (d). Calibration 5 μm.

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4. Fig. 3. Immunochemical staining of neurons expressing EGFP and fEGFP with antibodies to synaptic proteins. a, b – Fragments of dendrites of neurons expressing EGFP (a) and fEGFP (b) (green), stained with antibodies to the presynaptic protein synaptophysin (red). Arrows indicate some spines in which the head is adjacent to the immunopositive label. c, d – Neurons transfected with plasmids containing EGFP and fEGFP, stained with antibodies to the postsynaptic protein PSD95 (red). Arrows indicate coincidences of the immunoreactive label with the spine head. Calibration 5 μm.

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5. Fig. 4. Violin plots showing the distributions of the values ​​of various spine characteristics in neurons expressing EGFP and fEGFP. The thick dashed line shows the median, the dotted lines are the first quartiles. a – Spine length; b – the number of spines of a given morphological type or colocalized with the immunochemical label to the synaptic protein as a percentage of the total number of spines analyzed; right ordinate axis. * – p < 0.05; ** – p < 0.01; *** – p < 0.001; n.s. – differences are not significant, Student’s t-test with Holm–Bonferroni correction.

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