Additional file 1: of Rewiring neuronal microcircuits of the brain via spine head protrusions-a role for synaptopodin and intracellular calcium stores

SI Materials and Methods. (DOC 71 kb

Electrically evoked asynchronous EPSCs confirmed layer-specific increase in excitatory synaptic strength.

<p>(<b>A</b>) Asynchronous EPSCs (aEPSC) were recorded from identified dentate granule cells in control and denervated cultures upon local electrical stimulation of the OML and IML (recordings performed in Sr²⁺-containing bath solution; red, pipette containing the stimulating electrode and Alexa568; green, patch-clamp electrode containing Alexa488). Scale bar: 40 µm. (<b>B</b>) An increase in mean aEPSC amplitude was observed in response to electrical stimulations of the OML but not in response to electrical stimulations of the IML of denervated dentate granule cells (n = 4 cultures each; 1–3 neurons per culture).</p

Additional file 1: of Rewiring neuronal microcircuits of the brain via spine head protrusions-a role for synaptopodin and intracellular calcium stores

SI Materials and Methods. (DOC 71 kb

Entorhinal denervation in vitro leads to a layer-specific loss of excitatory input.

<p>(<b>A</b>) Schematic of an organotypic entorhino-hippocampal slice culture. The entorhino-hippocampal fiber tract (red) terminating in the outer molecular layer (OML) of the dentate gyrus (DG) and the plane of transection (blue) are illustrated. (EC, entorhinal cortex; IML, inner molecular layer). (<b>B</b>) Control and denervated cultures (blue, TO-PRO nuclear stain; white, plane of transection). In all denervation experiments the EC was removed. This procedure does not damage the DG. Scale bar: 500 µm. (<b>C</b>) Electrical stimulations of the entorhinal cortex while recording evoked EPSCs from dentate granule cells revealed an intact and functional entorhino-hippocampal projection. The evoked EPSCs were blocked by application of the AMPA-receptor antagonist CNQX (10 µM). Scale bar: 500 µm. (<b>D</b>) Entorhinal denervation leads to a layer-specific loss of excitatory input in the OML, while dendritic segments in the IML are not denervated (red, Mini-Rubi traced entorhinal fibers; green, schematic of a dentate granule cell). Scale bar: 50 µm.</p

Additional file 2: of Rewiring neuronal microcircuits of the brain via spine head protrusions-a role for synaptopodin and intracellular calcium stores

SI Figure Legend. Figure S1 CA1 neurons in slices cultures from synaptopodin-knockout (SP-KO) mice have comparable morphological and functional properties to wild type (WT) slices. a, Examples of CA1 dendrites rendered in 3D from WT and SP-KO slices. Scale bar, 2 μm. b, Quantification of spine densities, lengths and volumes from WT and SP-KO slices, expressed as percent of WT values. For spine densities, WT, n = 23 branches, 599 μm of dendrite from 22 slices; SP-KO, n = 23 branches, 645 μm of dendrite from 18 slices were analyzed. For spine lengths and volumes, WT, n = 1,021 spines from 22 slices; SP-KO, n = 1,121 spines from 18 slices were studied. c, Cumulative probability distributions of spine lengths (left) and spine volumes (right) in WT and SP-KO slice cultures. d, Example traces of mEPSCs recorded from CA1 pyramidal cells in WT (left) and SP-KO (right) slice cultures. e, Quantification of mean mEPSC amplitude (left; WT, 13.9 ± 0.38 pA and SP-KO, 13.6 ± 0.67 pA) and mean inter-mEPSC interval (right; WT, 228.37 ± 25.97 ms; SP-KO, 328.06 ± 49.14 ms). WT, n = 17 cells from 10 cultures; SP-KO, n = 15 cells from 8 cultures. f, Cumulative probability distributions of mEPSC amplitudes (left) and inter-mEPSC intervals (right) from WT and SP-KO CA1 neurons. (TIF 360 kb

table_1_Denervated mouse CA1 pyramidal neurons express homeostatic synaptic plasticity following entorhinal cortex lesion.xlsx

Structural, functional, and molecular reorganization of denervated neural networks is often observed in neurological conditions. The loss of input is accompanied by homeostatic synaptic adaptations, which can affect the reorganization process. A major challenge of denervation-induced homeostatic plasticity operating in complex neural networks is the specialization of neuronal inputs. It remains unclear whether neurons respond similarly to the loss of distinct inputs. Here, we used in vitro entorhinal cortex lesion (ECL) and Schaffer collateral lesion (SCL) in mouse organotypic entorhino-hippocampal tissue cultures to study denervation-induced plasticity of CA1 pyramidal neurons. We observed microglia accumulation, presynaptic bouton degeneration, and a reduction in dendritic spine numbers in the denervated layers 3 days after SCL and ECL. Transcriptome analysis of the CA1 region revealed complex changes in differential gene expression following SCL and ECL compared to non-lesioned controls with a specific enrichment of differentially expressed synapse-related genes observed after ECL. Consistent with this finding, denervation-induced homeostatic plasticity of excitatory synapses was observed 3 days after ECL but not after SCL. Chemogenetic silencing of the EC but not CA3 confirmed the pathway-specific induction of homeostatic synaptic plasticity in CA1. Additionally, increased RNA oxidation was observed after SCL and ECL. These results reveal important commonalities and differences between distinct pathway lesions and demonstrate a pathway-specific induction of denervation-induced homeostatic synaptic plasticity.</p

table_3_Denervated mouse CA1 pyramidal neurons express homeostatic synaptic plasticity following entorhinal cortex lesion.xlsx

Structural, functional, and molecular reorganization of denervated neural networks is often observed in neurological conditions. The loss of input is accompanied by homeostatic synaptic adaptations, which can affect the reorganization process. A major challenge of denervation-induced homeostatic plasticity operating in complex neural networks is the specialization of neuronal inputs. It remains unclear whether neurons respond similarly to the loss of distinct inputs. Here, we used in vitro entorhinal cortex lesion (ECL) and Schaffer collateral lesion (SCL) in mouse organotypic entorhino-hippocampal tissue cultures to study denervation-induced plasticity of CA1 pyramidal neurons. We observed microglia accumulation, presynaptic bouton degeneration, and a reduction in dendritic spine numbers in the denervated layers 3 days after SCL and ECL. Transcriptome analysis of the CA1 region revealed complex changes in differential gene expression following SCL and ECL compared to non-lesioned controls with a specific enrichment of differentially expressed synapse-related genes observed after ECL. Consistent with this finding, denervation-induced homeostatic plasticity of excitatory synapses was observed 3 days after ECL but not after SCL. Chemogenetic silencing of the EC but not CA3 confirmed the pathway-specific induction of homeostatic synaptic plasticity in CA1. Additionally, increased RNA oxidation was observed after SCL and ECL. These results reveal important commonalities and differences between distinct pathway lesions and demonstrate a pathway-specific induction of denervation-induced homeostatic synaptic plasticity.</p

table_4_Denervated mouse CA1 pyramidal neurons express homeostatic synaptic plasticity following entorhinal cortex lesion.xlsx

Structural, functional, and molecular reorganization of denervated neural networks is often observed in neurological conditions. The loss of input is accompanied by homeostatic synaptic adaptations, which can affect the reorganization process. A major challenge of denervation-induced homeostatic plasticity operating in complex neural networks is the specialization of neuronal inputs. It remains unclear whether neurons respond similarly to the loss of distinct inputs. Here, we used in vitro entorhinal cortex lesion (ECL) and Schaffer collateral lesion (SCL) in mouse organotypic entorhino-hippocampal tissue cultures to study denervation-induced plasticity of CA1 pyramidal neurons. We observed microglia accumulation, presynaptic bouton degeneration, and a reduction in dendritic spine numbers in the denervated layers 3 days after SCL and ECL. Transcriptome analysis of the CA1 region revealed complex changes in differential gene expression following SCL and ECL compared to non-lesioned controls with a specific enrichment of differentially expressed synapse-related genes observed after ECL. Consistent with this finding, denervation-induced homeostatic plasticity of excitatory synapses was observed 3 days after ECL but not after SCL. Chemogenetic silencing of the EC but not CA3 confirmed the pathway-specific induction of homeostatic synaptic plasticity in CA1. Additionally, increased RNA oxidation was observed after SCL and ECL. These results reveal important commonalities and differences between distinct pathway lesions and demonstrate a pathway-specific induction of denervation-induced homeostatic synaptic plasticity.</p

image_1_v1_Denervated mouse CA1 pyramidal neurons express homeostatic synaptic plasticity following entorhinal cortex lesion.tif

Structural, functional, and molecular reorganization of denervated neural networks is often observed in neurological conditions. The loss of input is accompanied by homeostatic synaptic adaptations, which can affect the reorganization process. A major challenge of denervation-induced homeostatic plasticity operating in complex neural networks is the specialization of neuronal inputs. It remains unclear whether neurons respond similarly to the loss of distinct inputs. Here, we used in vitro entorhinal cortex lesion (ECL) and Schaffer collateral lesion (SCL) in mouse organotypic entorhino-hippocampal tissue cultures to study denervation-induced plasticity of CA1 pyramidal neurons. We observed microglia accumulation, presynaptic bouton degeneration, and a reduction in dendritic spine numbers in the denervated layers 3 days after SCL and ECL. Transcriptome analysis of the CA1 region revealed complex changes in differential gene expression following SCL and ECL compared to non-lesioned controls with a specific enrichment of differentially expressed synapse-related genes observed after ECL. Consistent with this finding, denervation-induced homeostatic plasticity of excitatory synapses was observed 3 days after ECL but not after SCL. Chemogenetic silencing of the EC but not CA3 confirmed the pathway-specific induction of homeostatic synaptic plasticity in CA1. Additionally, increased RNA oxidation was observed after SCL and ECL. These results reveal important commonalities and differences between distinct pathway lesions and demonstrate a pathway-specific induction of denervation-induced homeostatic synaptic plasticity.</p

table_5_Denervated mouse CA1 pyramidal neurons express homeostatic synaptic plasticity following entorhinal cortex lesion.xlsx

Structural, functional, and molecular reorganization of denervated neural networks is often observed in neurological conditions. The loss of input is accompanied by homeostatic synaptic adaptations, which can affect the reorganization process. A major challenge of denervation-induced homeostatic plasticity operating in complex neural networks is the specialization of neuronal inputs. It remains unclear whether neurons respond similarly to the loss of distinct inputs. Here, we used in vitro entorhinal cortex lesion (ECL) and Schaffer collateral lesion (SCL) in mouse organotypic entorhino-hippocampal tissue cultures to study denervation-induced plasticity of CA1 pyramidal neurons. We observed microglia accumulation, presynaptic bouton degeneration, and a reduction in dendritic spine numbers in the denervated layers 3 days after SCL and ECL. Transcriptome analysis of the CA1 region revealed complex changes in differential gene expression following SCL and ECL compared to non-lesioned controls with a specific enrichment of differentially expressed synapse-related genes observed after ECL. Consistent with this finding, denervation-induced homeostatic plasticity of excitatory synapses was observed 3 days after ECL but not after SCL. Chemogenetic silencing of the EC but not CA3 confirmed the pathway-specific induction of homeostatic synaptic plasticity in CA1. Additionally, increased RNA oxidation was observed after SCL and ECL. These results reveal important commonalities and differences between distinct pathway lesions and demonstrate a pathway-specific induction of denervation-induced homeostatic synaptic plasticity.</p