DOI: 10.5061/dryad.v15dv420w
title: Denervated mouse CA1 pyramidal neurons express homeostatic synaptic plasticity following entorhinal cortex lesion

description: 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 three 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 three 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. 
description: <strong>Ethics statement </strong>
 Mice were maintained in a 12-hour light/dark cycle with food
 and water available <em>ad libitum</em>. Every effort was made
 to minimize distress and pain of animals. All experimental procedures were
 performed according to the German animal welfare legislation and approved
 by the animal welfare committee and/or the animal welfare officer at the
 Albert-Ludwigs-University Freiburg, Faculty of Medicine (X-17/07K,
 X-18/02C, X-21/01B).  <strong>Preparation of organotypic tissue
 cultures </strong> Entorhino-hippocampal
 tissue cultures were prepared at postnatal day 4–5 from
 C57BL/6J<em> </em>and Thy1-eGFP animals of either sex as
 previously described. Cultivation medium contained 50 % (v/v) MEM, 25 %
 (v/v) basal medium eagle, 25 % (v/v) heat-inactivated normal horse serum,
 25 mM HEPES buffer solution, 0.15 % (w/v) bicarbonate, 0.65 % (w/v)
 glucose, 0.1 mg ml<sup>-1</sup> streptomycin, 100 U
 ml<sup>-1</sup> penicillin, and 2 mM glutamax. The pH was
 adjusted to 7.3 and the medium was replaced three times per week. All
 tissue cultures were allowed to mature for at least 18 days in a
 humidified atmosphere with 5 % CO<sub>2</sub> at 35 °C, since
 at this age a steady-state in structural and functional properties of the
 organotypic tissue cultures is reached. <strong>Mechanical pathway
 lesion</strong> Mechanical pathway
 transection was performed with a sterile scalpel in mature tissue cultures
 (≥ 18 days<em> in vitro</em>). To apply an entorhinal cortex
 lesion (ECL), the perforant path was transected from the rhinal to the
 hippocampal fissure (Figure 1A). Schaffer collateral lesion (SCL) was
 applied between the CA3 and the CA1 region of the hippocampus, without
 affecting the perforant path projections to CA3 (Figure 1A). Except for
 the lesion-induced partial denervation of CA1 pyramidal neurons,
 cytoarchitecture of both the hippocampus and the entorhinal cortex
 remained unchanged. <strong>Chemogenetic pathway
 silencing</strong> pAAV-hSyn-hM4D(Gi)-mCherry was a gift from Bryan Roth
 (Addgene viral prep #-50475-AAV2; http://n2t.net/addgene:50475; RRID: Addgene_50475; 7 x 10<sup>12</sup> vg/ml, 1:4 diluted in PBS). AAV was injected into the CA3 region or the entorhinal cortex at 3 – 5 days<em> in vitro</em> using borosilicate glass pipettes. Cultures were returned to the incubator immediately after injection and allowed to mature for at least 18 days in a humidified atmosphere with 5 % CO<sub>2</sub> at 35 °C. Silencing was achieved by clozapine <em>N</em>-oxide treatment (CNO; 100 µM, 2 days; Tocris, #4936). Vehicle-only treatment (0.1% (v/v) DMSO) served as control in these experiments. <strong>Immunohistochemistry</strong> Cultures were fixed in a solution of 4 % (w/v) paraformaldehyde (PFA) in phosphate-buffered saline (PBS, 0.1 M, pH 7.4) and 4 % (w/v) sucrose for 1 h. Fixed cultures were incubated for 1 h with 10 % (v/v) normal goat serum (NGS) in 0.5 % (v/v) Triton X-100-containing PBS to block non-specific staining. Whole tissue cultures were incubated with rabbit anti-Iba1 (1:1000; Fujifilm Wako, #019-19741) or mouse anti-DNA/RNA oxidative damage (8-hydroxyguanosine (oh8G); 1:1000; QED Bioscience, #12501), in PBS containing 10 % (v/v) normal goat serum (NGS) and 0.1 % (v/v) Triton X-100 at 4°C overnight. Cultures were washed and incubated for 3 h with appropriate secondary antibodies (1:1000, in PBS with 10 % NGS or NHS, 0.1 % Triton X-100; Invitrogen). DAPI nuclear stain (1:5000 in PBS for 10 min; Thermo Scientific, #62248) was used to visualize cytoarchitecture. Sections were washed, transferred onto glass slides, and mounted for visualization with anti-fading mounting medium (DAKO Fluoromount).   Confocal images in immunostainings were acquired using a Leica SP8 confocal microscope equipped with a 20x (NA 0.75, Leica) or 40x (NA 1.3, Leica) objective lens. Detector gain and amplifier were initially set to obtain pixel intensities within a linear range. <strong>Posthoc-staining</strong> Cultures were fixed in a solution of 4 % (w/v) paraformaldehyde (PFA) in phosphate-buffered saline (PBS, 0.1 M, pH 7.4) and 4 % (w/v) sucrose for 1 h. Fixed cultures were incubated for 1 h with 10 % (v/v) normal goat serum (NGS) in 0.5 % (v/v) Triton X-100-containing PBS. Biocytin-filled cells were counterstained with Alexa 647-conjugated streptavidin (1:1000 in PBS with 10 % NGS, 0.1 % Triton X-100; Invitrogen, #S-32357) for 4 h and DAPI staining was used to visualize cytoarchitecture (1:5000 in PBS for 10 min; Thermo Scientific, #62248). Slices were washed, transferred, and mounted onto glass slides for visualization with anti-fading mounting medium (DAKO Fluoromount). Confocal images were acquired using a Leica SP8 confocal microscope equipped with a 20x objective lens (NA 0.75, Leica).  <strong>Transmission Electron Microscopy</strong> Tissue cultures were fixed in 4 % paraformaldehyde (w/v) and 2 % glutaraldehyde (w/v) in 0.1 M phosphate buffer (PB) overnight and washed for 1 hour in 0.1 M PB. After fixation, tissue cultures were incubated with 1 % osmium tetroxide for 20 min in 5 % (w/v) sucrose containing 0.1 M PB. The slices were washed 5 times for 10 min in 0.1 M PB and washed in graded ethanol (10 min in 10 % (v/v) and 10 min in 20 (v/v)). The slices were then incubated with uranyl acetate (1 % (w/v) in 70 % (v/v) ethanol) overnight and subsequently dehydrated in graded ethanol 80 % (v/v), 90 % (v/v) and 98 % (v/v) for 10 min. Finally, slices were incubated with 100 % (v/v) ethanol two times for 15 min followed by two 15 min washes with propylene oxide. The slices were then transferred for 30 min in a 1:1 mixture of propylene oxide with durcupan and then for 1 hour in durcupan. The durcupan was exchanged for fresh durcupan and the slices were transferred to 4 °C overnight. The slices were then embedded between liquid release-coated slides and coverslips. Cultures were re-embedded in blocks and ultrathin sections were collected on copper grids. Electron microscopy was performed with a LEO 906E microscope (Zeiss) at 3596x magnification. Acquired images were saved as TIF files and analyzed using the ImageSP Viewer software (http://e.informer.com/sys-prog.com). Asymmetric spine synapses were identified and manually quantified by an investigator blind to experimental conditions and hypotheses.   <strong>Whole-cell patch-clamp recordings of excitatory neurotransmission</strong> Whole-cell voltage-clamp recordings from CA1 pyramidal neurons of slice cultures were carried out at 35 °C (2-5 neurons per culture). The bath solution contained 126 mM NaCl, 2.5 mM KCl, 26 mM NaHCO<sub>3</sub>, 1.25 mM NaH<sub>2</sub>PO<sub>4</sub>, 2 mM CaCl<sub>2</sub>, 2 mM MgCl<sub>2</sub>, and 10 mM glucose. For EPSC recordings patch pipettes contained 126 mM K-gluconate, 4 mM KCl, 4 mM Mg-ATP, 0.3 mM Na<sub>2</sub>-GTP, 10 mM phosphocreatine, 10 mM HEPES and 0.3 % (w/v) biocytin (pH = 7.25 with KOH, 290 mOsm with sucrose) having a tip resistance of 4-6 MΩ. Cells were visually identified using an LN-Scope (Luigs and Neumann, Ratingen, Germany) equipped with infrared dot-contrast and a 40x water-immersion objective (NA 0.8, Olympus). Electrophysiological signals were amplified using a Multiclamp 700B amplifier, digitized with a Digidata 1550B digitizer, and visualized with the pClamp 11 software package. Spontaneous excitatory postsynaptic currents (sEPSCs) of CA1 pyramidal neurons were recorded in voltage-clamp mode at a holding potential of -60 mV. In chemogenetic silencing experiments, miniatures EPSCs (mEPSCs) were recorded to avoid an increase of synaptic activity in silenced pathways. For mEPSC recordings, D-APV (10 µM; Abcam, #ab120003), tetrodotoxin (TTX, 0.5 µM; Biotrend, #18660-81-6) and bicuculline-methiodide (10 µM; Abcam, #ab120108) were added to the external solution and the holding potential was set to -70 mV. Series resistance was monitored before and after each recording and recordings were discarded if the series resistance reached ≥ 30 MΩ.   <strong>Regional mRNA library preparation and transcriptome analysis</strong> RNA library preparations for transcriptome analysis were performed using the NEBNext<sup>®</sup> Single Cell/Low Input RNA Library Prep Kit for Illumina<sup>®</sup> (New England Biolabs, #E6420) according to the manufacturer’s instructions. Briefly, isolation of the CA1 region from individual tissue cultures was performed from non-lesioned control and SCL and ECL cultures three days after the lesion using a scalpel without collecting the scar tissue at the lesion site.  The tissue of one single isolated CA1 region was transferred to 7.5 µl lysis buffer (supplemented with murine RNase inhibitor) and homogenized using a pestill. Samples were centrifuged for 30 seconds at 10000g and 5 µl of supernatant were collected from individual samples and further processed. After cDNA synthesis, cDNA amplification was performed according to the manufacturer’s protocol. The cDNA yield was subsequently analyzed by a High Sensitivity DNA assay on a Bioanalyzer instrument (Agilent). The amount of cDNA was adjusted to 10 ng for further downstream applications. After fragmentation and adaptor ligation, dual index primers (New England Biolabs, #E7600S) were ligated in a library amplification step using 10 PCR cycles. Libraries were finally cleaned up with 0.8X SPRI beads following a standard bead purification protocol. Library purity and size distribution were assessed with a High Sensitivity DNA assay on a Bioanalyzer instrument (Agilent). We quantified the libraries using the NEBNext Library Quant Kit for Illumina (New England Biolabs, #E7630) based on the mean insert size provided by the Bioanalyzer. A 10 nM sequencing pool (120 µl in Tris-HCl, pH 8.5) was generated for sequencing on the NovaSeq6000 sequencing platform (Illumina; service provided by CeGaT GmbH, Tübingen, Germany). We performed a paired-end sequencing with 150 bp read length. Data analysis was performed at the Galaxy platform (usegalaxy.eu). All files contained more than 10 M high-quality reads (after mapping to the reference genome mm10) with a phred quality of at least 30 (&gt;90% of total reads). <strong>Time-lapse imaging of dendritic spines in CA1 pyramidal neurons</strong> Time-lapse imaging of dendritic spines was performed in Thy1-eGFP tissue cultures at a Zeiss LSM800 microscope equipped with a 10x (NA 0.3; Carl Zeiss) and a 60x objective (NA 1.0; Carl Zeiss). Filter membranes with 3-6 cultures were placed in a 35 mm Petri Dish containing pre-warmed and -oxygenated imaging solution consisting of 50% (v/v) MEM, 25% (v/v) BME, 50 mM HEPES buffer solution (25%, v/v), 0.65% (w/v) glucose, 0.15% (w/v) bicarbonate, 0.1 mg/ml streptomycin, 100 U/ml penicillin, 2 mM glutamax, and 0.1 mM trolox. The cultures were kept at 35°C during the imaging procedure. Baseline imaging was performed immediately before applying SCL or ECL respectively. Equally handled non-lesioned cultures served as control cultures.  Laser intensity and detector gain were initially set to keep the fluorescent signal in a dynamic range throughout the experiment and were kept constant during time series. For each dendritic layer in the CA1 region, a z-stack containing multiple dendritic segments was recorded before lesion (day 0) with Δz = 0.4 µm at ideal Nyquist rate and an optical zoom of 1. After imaging, lesions were applied and cultures were returned to the incubator. The imaging procedure was repeated 3 days after the lesion (day 3) following the same experimental protocol with the same imaging parameters. Confocal image stacks were stored as .czi files. <strong>Quantification and Statistics</strong> In this study, we used age-matched organotypic entorhino-hippocampal tissue cultures of either sex in a prospective study design to elucidate SCL- and ECL-induced plasticity in the CA1 region with non-lesioned cultures serving as controls. Electrophysiological data were analyzed using pClamp 10.7 (Axon Instruments) software. EPSC properties were analyzed using the automated template search tool for event detection. Microglial density was assessed by counting immunostained microglia manually in defined dendritic layers. The fluorescence signal of immunostained oxRNA was assessed in dendritic layers of the CA1 region in single-plane images. To avoid the detection of somatic signals, the regions of interest (ROIs) were placed in areas that did not contain any nuclei or somata. All analyses using immunohistochemistry were performed by investigators blind to experimental conditions. Dendritic spine density was determined by the automated detection tool ‘Spine Density Counter’ (DOI: 10.5281/zenodo.6712248) in the Fiji software environment. Z-stacked fluorescent images were projected at maximum intensity to create a 2D representation of individual dendritic segments. ImageJ plugin ‘Spine Density Counter’ was used to detect spines, count spine numbers, measure segment length and to subsequently calculate spine density. For one dendritic segment imaged at different time points, special attention was paid to ensure that the same starting and ending points at the respective segment were used; the same pixel resolution was applied in the algorithm for spine detection in all images. Posthoc visual inspection was applied to ensure the spine detection results were not strongly biased. Both raw spine density and normalized spine density to baseline were used in the analysis. Synaptic degeneration was analyzed in electron micrographs of the dendritic layers in the CA1 region. Membrane disintegration, swelling and the extensive loss of synaptic vesicles were considered as signs for degeneration. Analysis was performed manually by an investigator blind to experimental conditions. RNA sequencing data were uploaded to the galaxy web platform (public server: usegalaxy.eu) and transcriptome analysis was performed using the Galaxy platform in accordance with the reference-based RNA-seq data analysis tutorial. Adapter sequences, low quality, and short reads were removed via the CUTADAPT tool (Galaxy version 3.5+galaxy0). Reads were mapped using RNA STAR (Galaxy version 2.7.8a+galaxy0) with the mm10 full reference genome (<em>Mus</em> <em>musculus</em>). The evidence-based annotation of the mouse genome (GRCm38), version M25 (Ensembl 100) served as gene model (GENCODE). For an initial assessment of gene expression, unstranded FEATURECOUNT (Galaxy version 2.0.1+galaxy2) analysis was performed from RNA STAR output. Only samples that contained &gt;60% uniquely mapping reads (feature: “exon”) were considered for further analysis. Genes with a low number of mean reads (base mean &lt; 150 counts) were excluded from further analysis.  Read counts were further analyzed using DESeq2. The functional enrichment analysis was performed using g:Profiler (version e107_eg54_p17_bf42210) with g:SCS multiple testing correction method applying significance threshold of 0.05. Gene sets with 50–500 terms were considered for illustration. Data were statistically analyzed using GraphPad Prism 9 (GraphPad software, USA). For statistical comparison of two unpaired experimental groups, a Mann-Whitney test was applied. For statistical comparison of two paired experimental groups (time-lapse imaging data sets), we used the Wilcoxon matched-pair signed rank test in normalized and non-normalized data. For the evaluation of data sets with three experimental groups, a Kruskal-Wallis test followed by Dunn’s posthoc correction was applied. Amplitude/frequency plots were statistically assessed by the repeated measure (RM) two-way ANOVA test with Sidak’s (two groups) multiple comparisons test. mEPSC frequency values from individual cells were stacked in subcolumns and mEPSC amplitude bins defined tabular rows (COLUMN factor: treatment; ROW factor: amplitude bin). P-values &lt; 0.05 were considered statistically significant (*p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001). The n-numbers are provided in the figure legends. Results that did not yield significant differences were designated ‘ns’. Statistical differences from RM-two-way ANOVA test in XY-plots were indicated in the legend of the figure panels (*) when detected through multiple comparisons. In the text and figures, values represent the mean ± standard error of the mean (s.e.m.)  <strong>Data and material availability</strong> Source data with statistical evaluations for each figure are provided here. Raw data (.fastq-files) used for transcriptome analysis are available at the Gene Expression Omnibus; accession number GSE223096. Original data are available from the corresponding authors upon reasonable request. <strong>Digital illustrations</strong> Confocal image stacks were stored as TIF files. Figures were prepared using the ImageJ software package (https://imagej.nih.gov/ij/) and Photoshop graphics software (Adobe, San Jose, CA, USA). Image brightness and contrast were adjusted. 
description: Data were statistically analyzed using GraphPad Prism 9 (GraphPad
 software, USA).