DOI: 10.5061/dryad.x0k6djhpc
title: Synaptopodin regulates denervation-induced plasticity at hippocampal mossy fiber synapses

description: Neurological diseases can lead to the denervation of brain regions caused
 by demyelination, traumatic injury or cell death. The molecular and
 structural mechanisms underlying lesion-induced reorganization of
 denervated brain regions, however, are a matter of ongoing investigation.
 In order to address this issue, we performed an entorhinal cortex lesion
 (ECL) in mouse organotypic entorhino-hippocampal tissue cultures of both
 sexes and studied denervation-induced plasticity of mossy fiber synapses,
 which connect dentate granule cells (dGCs) with CA3 pyramidal cells
 (CA3-PCs) and play important roles in learning and memory formation.
 Partial denervation caused a strengthening of excitatory neurotransmission
 in dGCs, CA3-PCs and their direct synaptic connections, as revealed by
 paired recordings (dGC-to-CA3-PC). These functional changes were
 accompanied by ultrastructural reorganization of mossy fiber synapses,
 which regularly contain the plasticity-regulating protein synaptopodin and
 the spine apparatus organelle. We demonstrate that the spine apparatus
 organelle and synaptopodin are related to ribosomes in close proximity to
 synaptic sites and unravel a synaptopodin-related transcriptome. Notably,
 synaptopodin-deficient tissue preparations that lack the spine apparatus
 organelle failed to express lesion-induced synaptic adjustments. Hence,
 synaptopodin and the spine apparatus organelle play a crucial role in
 regulating lesion-induced synaptic plasticity at hippocampal mossy fiber
 synapses.
description: <strong>Ethics statement</strong>
 Mice were maintained in a 12 h light/dark cycle with food and
 water available ad libitum. Every effort was made to minimize distress and
 pain of animals. All experimental procedures were performed according to
 German animal welfare legislation and approved by the appropriate animal
 welfare committee and the animal welfare officer of
 Albert-Ludwigs-University Freiburg, Faculty of Medicine (X-17/07K,
 X-17/09K, X-21/01B) and Hannover Medical School (2022/308).
 <strong>Preparation of tissue
 cultures</strong> Entorhino-hippocampal tissue
 cultures were prepared at postnatal day 3-5 from C57BL/6J (wildtype,
 Synpo<sup>+/+</sup>) and
 B6.129-Synpo<sup>tm1Mndl/Dllr</sup>
 (Synpo<sup>-/-</sup>) mice of either sex.
 Synpo<sup>-/-</sup> strain was back crossed for at least 10
 times to establish C57BL/6J genetic background. 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 streptomycin, 100 U/ml
 penicillin, and 2 mM glutamax. The pH was adjusted to 7.3. 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. Cultivation medium
 was replaced 3 times per week. <strong>Entorhinal
 cortex lesion (ECL)</strong> The entorhinal
 cortex of mature slice cultures (≥18 days <em>in vitro</em>)
 was transected with a sterile scalpel from the rhinal to the hippocampal
 fissure to achieve a complete lesion of the perforant path. Except for the
 lesion-induced partial denervation of the hippocampus, cytoarchitecture of
 both the hippocampus and the entorhinal cortex remained
 unchanged. <strong>Whole-cell patch-clamp
 recordings</strong> Whole-cell patch-clamp
 recordings were carried out at 35°C (3-6 neurons per culture). The bath
 solution contained (in mM) 126 NaCl, 2.5 KCl, 26
 NaHCO<sub>3</sub>, 1.25
 NaH<sub>2</sub>PO<sub>4</sub>, 2
 CaCl<sub>2</sub>, 2 MgCl<sub>2</sub>, and 10
 glucose (aCSF) and was oxygenated continuously (5%
 CO<sub>2</sub> / 95% O<sub>2</sub>). Patch
 pipettes contained (in mM) 126 K-gluconate, 10 HEPES, 4 KCl, 4 ATP-Mg, 0.3
 GTP-Na<sub>2</sub>, 10 PO-Creatine, 0.3% (w/v) biocytin (pH
 7.25 with KOH, 290 mOsm with sucrose), having a tip resistance of 4-6 MΩ.
 Neurons were visually identified using a LN-Scope (Luigs &
 Neumann) equipped with an infrared dot-contrast and a 40x water-immersion
 objective (NA 0.8; Olympus). Spontaneous excitatory postsynaptic currents
 (sEPSCs) of both dGCs and CA3-PCs were recorded in voltage-clamp mode at a
 holding potential of −70 mV. In the dentate gyrus, recordings were
 performed in granule cells at the outer parts of the granule cell layer in
 the suprapyramidal blade with maximum distance to the subgranular zone.
 Moreover, the resting membrane potential was estimated before our
 recordings to exclude immature granule cells. In CA3, recordings were
 performed in pyramidal cells close to the dentate gyrus in proximity to
 str. lucidum. Series resistance was monitored before and after each
 recording and recordings were discarded if the series resistance reached ≥
 30 MΩ. For recording of intrinsic cellular properties in current-clamp
 mode, pipette capacitance of 2.0 pF was corrected and series resistance
 was compensated using the automated bridge balance tool of the MultiClamp
 commander. I-V-curves were generated by injecting 1 s square pulse
 currents starting at −100 pA and increasing in 10 pA steps until +500 pA
 current injection was reached (sweep duration: 2 s).
 <strong>Paired whole-cell patch-clamp recordings of
 hippocampal mossy fiber synapses</strong> Action
 potentials were generated in the presynaptic dGC by 5 ms square current
 pulses (1 nA). For connectivity analysis, 5 action potentials were applied
 at 20 Hz respectively (inter-sweep-interval: 10 s; at least 30
 repetitions), while recording unitary excitatory postsynaptic currents
 (uEPSCs) from CA3-PCs. Neurons were considered to be connected if &gt;
 5 % of action potentials evoked time-locked inward uEPSCs. Recordings were
 performed in the presence of (-)-bicuculline-methiodide (10 µM, Abcam,
 #ab120108) to prevent inhibitory synapse recruitment. Moreover, glutamine
 (200 µM, Gibco, #11539876) was added to the recording medium to avoid
 synaptic depletion. <strong>Post-hoc staining and
 imaging</strong> Tissue cultures were fixed
 overnight in a solution of 4% (w/v) paraformaldehyde (PFA; in PBS (0.1M,
 pH 7.4) with 4% (w/v) sucrose). After fixation, cultures were washed in
 PBS (0.1 M, pH 7.4) and consecutively incubated for 1 h with 10% (v/v)
 normal goat serum (NGS) in 0.5% (v/v) Triton X-100 containing PBS to
 reduce nonspecific staining. For post-hoc visualization of patched dGCs
 and CA3-PCs, Streptavidin Alexa Fluor 488 (Streptavidin A488, 1:1000;
 Invitrogen, #S32354) was added to the incubation solution. DAPI nuclear
 stain (1:5000 in PBS for 10 min; Thermo Scientific, #62248) was used to
 visualize cytoarchitecture. Cultures were washed, transferred onto glass
 slides and mounted for visualization with DAKO anti-fading mounting medium
 (Agilent, #S302380-2). <strong>Serial block-face
 scanning electron microscopy (SBF-SEM)</strong>
 For volume electron microscopic evaluation, samples were fixed in
 1.5 % glutaraldehyde and 1.5 % paraformaldehyde buffered in 0.15 M HEPES,
 pH = 7.35. Further processing was based on accessible protocols
 (https://ncmir.ucsd.edu/sbem-protocol). In brief, for postfixation and
 staining reduced osmium tetroxide, thiocarbohydrazide and osmium tetroxide
 (rOTO), followed by uranyl acetate and lead aspartate were applied. After
 dehydration in an ascending acetone series, samples were embedded in epoxy
 resin, Araldite CY212 premix kit (Agar Scientific, Stansted Essex,
 UK). Target regions were identified on semithin
 sections (1 µm, stained with toluidine blue). Accordingly, resin blocks
 were trimmed, mounted with conductive epoxy glue (Chemtronics,
 CircuitWorks, Kennesaw, USA) on sample pin stubs (Micro to Nano, Haarlem,
 Netherlands), precisely trimmed again and sputter coated with a gold
 layer. Image stacks were acquired with a Zeiss Merlin
 VP Compact SEM (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) equipped
 with a Gatan 3View2XP system (Gatan Inc., Pleasanton, CA, USA). Every
 second block-face (section thickness 50 nm) was scanned with 3.5 kV
 acceleration voltage and 100 % focal charge compensation (10 nm pixel
 size, 3 µs dwell time) by using the Multi-ROI mode to record multiple
 areas on each surface. <strong>Transmission
 electron microscopy – mossy fiber synapse
 visualization</strong> Wildtype
 (Synpo<sup>+/+</sup>) and Synpo<sup>-/-</sup>
 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 sliced with a
 vibratome and the slices 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 were
 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
 twice 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 with 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, at which point an additional
 Pb-citrate contrasting step was performed (3 min). Electron microscopy was
 performed at a LEO 906E microscope (Zeiss) at 4646x
 magnification. <strong>Synaptopodin
 co-Immunoprecipitation (SP-coIP), RNA-Seq analysis and negative contrast
 electron microscopy</strong> Six tissue cultures
 per biological replicate were washed in coIP-solution and dounce
 homogenized on ice in a solution containing (150 µl per sample,
 coIP-solution, in mM): 10 HEPES, 120 NaCl, 0.05% (v/v) NP-40 or IGEPAL
 CA-630 (Sigma Aldrich, #I8896), cOmplete protease inhibitor (Roche,
 according to manufacturer’s instructions), murine RNase inhibitor (1:1000,
 New England Biolabs, #M0314) and cycloheximide (0.5 mg/ml, Sigma Aldrich,
 #C-4859). Input was precleared twice with protein A beads (10 µl per 100
 µl sample/supernatant) to reduce non-specific binding. Protein A beads (10
 µl, New England Biolabs, #S1425) were washed twice in coIP-solution and
 consecutively incubated for 1 hour at 4°C with an anti-synaptopodin
 antibody (rabbit anti-synaptopodin, Synaptic Systems, #163002). Beads were
 carefully washed twice to remove unbound antibodies and incubated for 1
 hour with the precleared supernatant (‘input’) at 4 °C with head-over-tail
 rotation. 10 µl of the precleared supernatant were kept as input control.
 After incubation, beads were washed twice with coIP-solution and finally
 dissolved in RNA protection buffer (New England Biolabs).
 In another round of experiments, hippocampal tissue from adult
 animals was used for co-immunoprecipitation experiments (antibody:
 monoclonal rabbit anti-synaptopodin, Abcam, #ab259976 Abcam) with
 subsequent negative contrast electron microscopy. Beads (30 µl) were
 adsorbed on carbon-coated films stabilized by a copper grid and stained
 with 3% uranyl acetate (Serva, Heidelberg, Germany). Samples were
 immediately examined with a transmission electron microscope (Morgagni
 268, 80 kV, FEI, Eindhoven, The Netherlands). RNA was
 released from the beads through protein K lysis
 (Monarch<sup>®</sup> Total RNA Miniprep Kit; New England
 Biolabs, #T2010S). After lysis, beads were removed from the solution in a
 magnetic rack and the RNA containing supernatant was transferred to fresh
 tubes. RNA isolation on coIP- and input-samples was then performed
 according to the manufacturer’s instructions. RNA content was determined
 using the Agilent RNA 6000 Pico Kit (Agilent, #5067-1513) with a 2100
 Bioanalyzer (Agilent, #G2939BA). RNA Library preparations for
 Synpo-related 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. 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) having at
 least a phred quality of 30 (&gt; 90 % of total reads).
 <strong>Regional mRNA library preparations 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 dentate gyrus and CA3 area from individual tissue cultures was
 performed using a scalpel. One isolated dentate gyrus or CA3 area
 respectively was transferred to 7.5 µl lysis buffer (supplemented with
 murine RNase inhibitor) and homogenized using a pestle. Samples were
 centrifuged for 30 s at 10,000 g 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
 with 12 PCR cycles. cDNA yield was consecutively analyzed by a High
 Sensitivity DNA assay on a Bioanalyzer instrument (Agilent). cDNA amount
 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 was 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 100 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) having at least a phred quality of 30
 (&gt;90% of total reads).
description: <strong>Experimental Design and Statistical Analysis
 </strong> Electrophysiological data were analyzed
 using pClamp 10.7 (Axon Instruments) and MiniAnalysis (Synaptosoft)
 software. The fraction of action potentials followed by time-locked
 excitatory postsynaptic current responses was considered as synaptic
 response rate. uEPSC amplitude was assessed in uEPSCs from successfully
 transmitted action potentials as well as the mean amplitude of all
 successfully evoked postsynaptic responses. In order to ensure analysis of
 time-locked unitary responses, events were included if &gt; 5% of
 presynaptic pulses were successfully transmitted for each pulse
 respectively. For individual pulses in pulse trains that caused ≤ 5%
 successfully transmitted events, response rate was set to ‘0’. sEPSC
 properties were analyzed using the automated template search tool for
 event detection. Since sEPSC kinetics might contain information on their
 synaptic origin (in CA3-PCs, large EPSCs have been identified to originate
 from hippocampal mossy fibers), we performed a hierarchical analysis of
 sEPSCs based on their amplitude. All events of a recorded cell were sorted
 based on their peak amplitude. Events above the
 50<sup>th</sup> percentile therefore correspond to all events
 above the cells’ median amplitude of events. Amplitude and half width data
 from sEPSC analysis have been plotted to visualize sEPSC kinetics and
 linear fitting was employed to detect changes in parameter
 interdependencies. sEPSC area was reported as a function of amplitude and
 half width. Input resistance was calculated for the injection of –100 pA
 current at a time frame of 200 ms with a maximum distance to the initial
 hyperpolarization. Resting membrane potential was calculated as the mean
 baseline value. AP detection was performed using the input-output curve
 threshold search event detection tool, and the AP frequency was assessed
 by the number of APs detected during the respective current injection
 time. 3D reconstructions of individual dendritic spines
 in electron micrograph image stacks were performed using the TrakEM2 tool
 in the Fiji software environment by 3 individual investigators blind to
 experimental conditions and hypothesis. From one image stack, subfields
 were selected (proximal to soma: dentate gyrus (DG), 10-50 µm; CA3, 30-90
 µm distance to soma; distal to soma: &gt;100 µm distance to soma in
 both subfields) and all pre- and postsynaptic compartments of asymmetric
 synapses that were fully captured on image stacks were manually
 reconstructed. In order to analyze synaptic complexity, the images with
 the 3D-reconstructed synaptic compartments were used. The number of
 postsynaptic compartments and their postsynaptic densities were manually
 counted for each respective presynaptic compartment by an investigator
 blinded to experimental conditions. Transmission
 electron microscopy images of mossy fiber synapses were saved as TIF-files
 and analyzed using the ImageSP Viewer software
 (http://e.informer.com/sys-prog.com). Ultrastructural features were
 analyzed by an investigator blind to experimental conditions in randomly
 selected mossy fiber terminals from electron micrographs of the stratum
 lucidum in non-lesioned and lesioned wildtype tissue cultures.
 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 sequencing, low quality, and short reads
 were removed via the CUTADAPT tool. Reads were mapped using RNA STAR with
 the mm10 full reference genome (Mus musculus). 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 analysis was performed from RNA STAR output. Only
 samples that contained &gt;60% uniquely mapping reads (feature:
 “exon”) were considered for further analysis. Statistical evaluation was
 performed using DESeq2 with “pathway integrity” (ECL experiments) or
 “coIP” (synaptopodin-coIP experiments) as the primary factor that might
 affect gene expression. Genes with a low number of mean reads were
 excluded from further analysis. Genes were considered as differentially
 expressed or enriched if the adjusted p-value was &lt; 0.05. Data
 visualization was performed according to a modified version of a
 previously published workflow. Further functional enrichment analyses were
 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 100-500 terms were considered for illustration.
 Heatmaps were generated based on z-scores of the normalized count
 table. For transcriptome analysis following
 synaptopodin-coIP, DESeq2 analysis was performed to compare coIP and input
 samples. Differentially enriched genes in coIP
 (p<sub>adj</sub> &lt; 0.05 and log2(FC) &gt; 0) were
 considered for further analyses. To correct for non-specific antibody
 binding, DESeq2 output (log2(FC)) from the same experimental procedure in
 synaptopodin-deficient tissue cultures was subtracted from log2(FC) of
 differentially enriched genes in wildtype cultures. Only those
 differentially expressed genes that showed &gt; 30% enrichment in FC
 after Synpo-KO correction were considered as significantly
 enriched. Data were statistically analyzed using
 GraphPad Prism 7 or 9 (GraphPad software, USA). For statistical comparison
 of two experimental groups, a Mann-Whitney test was employed. For
 statistical comparison of three experimental groups, a Kruskal-Wallis test
 was employed. In the graphs demonstrating volumes of the synaptic
 compartments, the box depicts 25-75 percentile, whiskers depict 10-90
 percentile and the line indicates the median. Values outside this range
 were indicated by individual dots. Otherwise, values represent the mean ±
 standard error of the mean (s.e.m.). sEPSC amplitude/frequency plots and
 AP-frequency plots were statistically assessed by the repeated measure
 (RM) two-way ANOVA test with Sidak’s (two groups) multiple comparisons
 test. uEPSC amplitude values from individual cells were stacked in
 subcolumns and the pulse number was defining tabular rows (COLUMN factor:
 pathway integrity, genetic background; ROW factor: EPSC amplitude bin or
 current injection). P-values &lt; 0.05 were considered statistically
 significant (*p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001);
 results that did not yield significant differences are designated ‘ns’.
 Statistical differences in XY-plots were indicated in the legend of the
 figure panels (*) when detected through multiple comparisons, irrespective
 of their localization and the level of significance.
 <strong>Data availability</strong>
 Sequencing data have been deposited in the Gene Expression
 Omnibus (GEO) repository (accession number: GSE216509). 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: # README for Synaptopodin regulates denervation-induced plasticity at
 hippocampal mossy fiber synapses Pia Kruse1, Gudrun Brandes2, Hanna
 Hemeling1, Zhong Huang2, Christoph Wrede3,4, Jan Hegermann3,4, Andreas
 Vlachos1,5,6, and Maximilian Lenz1,2, 1    Department of Neuroanatomy,
 Institute of Anatomy and Cell Biology, Faculty of Medicine, University of
 Freiburg, Freiburg, Germany. 2    Hannover Medical School, Institute of
 Neuroanatomy and Cell Biology, Hannover, Germany. 3    Hannover Medical
 School, Institute of Functional and Applied Anatomy, Hannover, Germany.
 4    Hannover Medical School, Research Core Unit Electron Microscopy,
 Hannover, Germany. 5    Center for Basics in Neuromodulation
 (NeuroModulBasics), Faculty of Medicine, University of Freiburg, Freiburg,
 Germany. 6    Center BrainLinks-BrainTools, University of Freiburg,
 Freiburg, Germany. Correspondence:
 and
 ## Description of the data and file
 structure #### General information Source Data for statistical analysis
 and data visualization. GraphPad files (GraphPad Prism software package)
 are structured according to the figures in the original manuscript.
 Abbreviations: DG: dentate gyrus ECL: entorhinal cortex lesion dGC:
 dentate granule cell PC: pyramidal cell STP: short term plasticity EPSC:
 excitatory postsynaptic current aCSF: artificial cerebrospinal fluid ROI:
 region of interest coIP: co-immunoprecipitation SA: spine apparatus
 organelle CC: cellular compartment MF: molecular function Area of
 investigations: Dentate gyrus (DG) and the CA3 subfield (CA3) in murine
 organotypic entorhino-hippocampal tissue cultures #### File structure of
 source data and statistical analysis Figure 1: GraphPad .pzfx-file;
 electrophysiological data from sEPSC recordings in both dGCs and CA3-PCs
 in BL6 tissue cultures Electrophysiological recordings: sEPSC amplitude
 [pA] Electrophysiological recordings: sEPSC half width [ms]
 Electrophysiological recordings: sEPSC area [pA x ms] Electrophysiological
 recordings: sEPSC frequency [Hz] Electrophysiological recordings: sEPSC
 amplitude [≤ 50. percentile; pA] Electrophysiological recordings: sEPSC
 amplitude [&gt; 50. percentile; pA] Electrophysiological recordings: XY
 correlation graph of amplitude and half width Electrophysiological
 recordings: amplitude/frequency correlation plot Figure 2: GraphPad
 .pzfx-file; electrophysiological data from I-V-curve recordings in both
 dGCs and CA3-PCs in BL6 tissue cultures Electrophysiological recordings:
 resting membrane potential [mV] Electrophysiological recordings: input
 resistance [MOhm] Electrophysiological recordings: I-V-curve
 Electrophysiological recordings: action potential frequency [Hz] Figure 3:
 GraphPad .pzfx-file; 3D electron microscopy data of distal and proximal
 synapses of both dGCs and CA3-PCs in BL6 tissue cultures 3D electron
 microscopy data: postsynaptic volume [µm^3] 3D electron microscopy data:
 presynaptic volume [µm^3] 3D electron microscopy data: number postsynaptic
 compartments per presynaptic compartment Figure 4: GraphPad .pzfx-file;
 electrophysiological data of paired whole-cell patch-clamp recordings
 (dGC-to-CA3-PC) in BL6 tissue cultures Electrophysiological recordings:
 response rate [fraction of total pulses] for pulse n°1-5
 Electrophysiological recordings: uEPSC amplitude [pA] for pulse n°1-5
 Figure 5: GraphPad .pzfx-file; transcriptome analyses, coIP-experiments,
 ultrastructural EM analyses Ultrastructural EM analyses: cross sections
 postsynaptic compartments and ribosomes [total] Ultrastructural EM
 analyses: cross sections postsynaptic compartments and ribosomes [no spine
 apparatus organelle] Ultrastructural EM analyses: cross sections
 postsynaptic compartments and ribosomes [spine apparatus organelle]
 Ultrastructural EM analyses: spine apparatus organelles [n° dense plates]
 Ultrastructural EM analyses: dense plate length [nm] Transcriptome
 analysis: Synpo-coIP enriched mRNA Figure 6: GraphPad .pzfx-file;
 electrophysiological data from sEPSC recordings and I-V-curves in both
 dGCs and CA3-PCs and of paired whole-cell patch-clamp recordings
 (dGC-to-CA3-PC) in Synpo-/- tissue cultures Electrophysiological
 recordings: sEPSC amplitude [pA] Electrophysiological recordings: sEPSC
 half width [ms] Electrophysiological recordings: sEPSC area [pA x ms]
 Electrophysiological recordings: sEPSC frequency [Hz] Electrophysiological
 recordings: XY correlation graph of amplitude and half width
 Electrophysiological recordings: amplitude/frequency correlation plot
 Electrophysiological recordings: resting membrane potential [mV]
 Electrophysiological recordings: I-V-curve Electrophysiological
 recordings: response rate [fraction of total pulses] for pulse n°1-5
 Electrophysiological recordings: uEPSC amplitude [pA] for pulse n°1-5
 Figure 7: GraphPad .pzfx-file; electrophysiological data from sEPSC
 recordings in CA3-PCs and of paired whole-cell patch-clamp recordings
 (dGC-to-CA3-PC) in BL6 and Synpo-/- tissue cultures Electrophysiological
 recordings: sEPSC amplitude [pA] Electrophysiological recordings: sEPSC
 half width [ms] Electrophysiological recordings: sEPSC area [pA x ms]
 Electrophysiological recordings: sEPSC frequency [Hz] Electrophysiological
 recordings: XY correlation graph of amplitude and half width
 Electrophysiological recordings: amplitude/frequency correlation plot
 Electrophysiological recordings: response rate [fraction of total pulses]
 for pulse n°1-5 Electrophysiological recordings: uEPSC amplitude [pA] for
 pulse n°1-5 Figure 8: GraphPad .pzfx-file; transcriptome analyses of both
 the dentate gyrus and CA3-region in BL6 and Synpo-/- tissue cultures
 Transcriptome analysis: differentially expressed genes Table 1: GraphPad
 .pzfx-file; 3D electron microscopy data of distal and proximal synapses of
 both dGCs and CA3-PCs in BL6 tissue cultures 3D electron microscopy data:
 PSD surface area [µm^2] ## Sharing/Access information Sequencing data have
 been deposited in the Gene Expression Omnibus (GEO) repository (accession
 number: GSE216509). Original data are available from the authors upon
 reasonable request. ## Methodological overview #### Preparation of tissue
 cultures Entorhino-hippocampal tissue cultures were prepared at postnatal
 day 3-5 from C57BL/6J (wildtype, Synpo+/+) and B6.129-Synpotm1Mndl/Dllr
 (Synpo-/-, [18-20]) mice of either sex as previously described. Synpo-/-
 strain was back crossed for at least 10 times to establish C57BL/6J
 genetic background. 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 streptomycin, 100 U/ml penicillin, and 2 mM glutamax. The pH was
 adjusted to 7.3. All tissue cultures were allowed to mature for at least
 18 days in a humidified atmosphere with 5% CO2 at 35°C. Cultivation medium
 was replaced 3 times per week. #### Entorhinal cortex lesion (ECL) The
 entorhinal cortex of mature slice cultures (≥18 days in vitro) was
 transected with a sterile scalpel from the rhinal to the hippocampal
 fissure to achieve a complete lesion of the perforant path. Except for the
 lesion-induced partial denervation of the hippocampus, cytoarchitecture of
 both the hippocampus and the entorhinal cortex remained unchanged. ####
 Whole-cell patch-clamp recordings Whole-cell patch-clamp recordings were
 carried out at 35°C (3-6 neurons per culture). The bath solution contained
 (in mM) 126 NaCl, 2.5 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 2 MgCl2, and
 10 glucose (aCSF) and was oxygenated continuously (5% CO2 / 95% O2). Patch
 pipettes contained (in mM) 126 K-gluconate, 10 HEPES, 4 KCl, 4 ATP-Mg, 0.3
 GTP-Na2, 10 PO-Creatine, 0.3% (w/v) biocytin (pH 7.25 with KOH, 290 mOsm
 with sucrose), having a tip resistance of 4-6 MΩ. Neurons were visually
 identified using a LN-Scope (Luigs & Neumann) equipped with an
 infrared dot-contrast and a 40x water-immersion objective (NA 0.8;
 Olympus). Spontaneous excitatory postsynaptic currents (sEPSCs) of both
 dGCs and CA3-PCs were recorded in voltage-clamp mode at a holding
 potential of −70 mV. In the dentate gyrus, recordings were performed in
 granule cells at the outer parts of the granule cell layer in the
 suprapyramidal blade with maximum distance to the subgranular zone.
 Moreover, the resting membrane potential was estimated before our
 recordings to exclude immature granule cells. In CA3, recordings were
 performed in pyramidal cells close to the dentate gyrus in proximity to
 str. lucidum. Series resistance was monitored before and after each
 recording and recordings were discarded if the series resistance reached ≥
 30 MΩ. For recording of intrinsic cellular properties in current-clamp
 mode, pipette capacitance of 2.0 pF was corrected and series resistance
 was compensated using the automated bridge balance tool of the MultiClamp
 commander. I-V-curves were generated by injecting 1 s square pulse
 currents starting at −100 pA and increasing in 10 pA steps until +500 pA
 current injection was reached (sweep duration: 2 s). #### Paired
 whole-cell patch-clamp recordings of hippocampal mossy fiber synapses
 Action potentials were generated in the presynaptic dGC by 5 ms square
 current pulses (1 nA). For connectivity analysis, 5 action potentials were
 applied at 20 Hz respectively (inter-sweep-interval: 10 s; at least 30
 repetitions), while recording unitary excitatory postsynaptic currents
 (uEPSCs) from CA3-PCs. Neurons were considered to be connected if &gt; 5 %
 of action potentials evoked time-locked inward uEPSCs. Recordings were
 performed in the presence of (-)-bicuculline-methiodide (10 µM, Abcam,
 #ab120108) to prevent inhibitory synapse recruitment. Moreover, glutamine
 (200 µM, Gibco, #11539876) was added to the recording medium to avoid
 synaptic depletion. #### Post-hoc staining and imaging Tissue cultures
 were fixed overnight in a solution of 4% (w/v) paraformaldehyde (PFA; in
 PBS (0.1M, pH 7.4) with 4% (w/v) sucrose). After fixation, cultures were
 washed in PBS (0.1 M, pH 7.4) and consecutively incubated for 1 h with 10%
 (v/v) normal goat serum (NGS) in 0.5% (v/v) Triton X-100 containing PBS to
 reduce nonspecific staining. For post-hoc visualization of patched dGCs
 and CA3-PCs, Streptavidin Alexa Fluor 488 (Streptavidin A488, 1:1000;
 Invitrogen, #S32354) was added to the incubation solution. DAPI nuclear
 stain (1:5000 in PBS for 10 min; Thermo Scientific, #62248) was used to
 visualize cytoarchitecture. Cultures were washed, transferred onto glass
 slides and mounted for visualization with DAKO anti-fading mounting medium
 (Agilent, #S302380-2). #### Serial block-face scanning electron microscopy
 (SBF-SEM) For volume electron microscopic evaluation, samples were fixed
 in 1.5 % glutaraldehyde and 1.5 % paraformaldehyde buffered in 0.15 M
 HEPES, pH = 7.35. Further processing was already described previously
 based on accessible protocols
 (). In brief, for postfixation
 and staining reduced osmium tetroxide, thiocarbohydrazide and osmium
 tetroxide (rOTO), followed by uranyl acetate and lead aspartate were
 applied. After dehydration in an ascending acetone series, samples were
 embedded in epoxy resin, Araldite CY212 premix kit (Agar Scientific,
 Stansted Essex, UK). Target regions were identified on semithin sections
 (1 µm, stained with toluidine blue). Accordingly, resin blocks were
 trimmed, mounted with conductive epoxy glue (Chemtronics, CircuitWorks,
 Kennesaw, USA) on sample pin stubs (Micro to Nano, Haarlem, Netherlands),
 precisely trimmed again and sputter coated with a gold layer. Image stacks
 were acquired with a Zeiss Merlin VP Compact SEM (Carl Zeiss Microscopy
 GmbH, Oberkochen, Germany) equipped with a Gatan 3View2XP system (Gatan
 Inc., Pleasanton, CA, USA). Every second block-face (section thickness 50
 nm) was scanned with 3.5 kV acceleration voltage and 100 % focal charge
 compensation (10 nm pixel size, 3 µs dwell time) by using the Multi-ROI
 mode to record multiple areas on each surface. #### Transmission electron
 microscopy – mossy fiber synapse visualization Wildtype (Synpo+/+) and
 Synpo-/- 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 sliced with a
 vibratome and the slices 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 were
 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
 twice 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 with 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, at which point an additional
 Pb-citrate contrasting step was performed (3 min). Electron microscopy was
 performed at a LEO 906E microscope (Zeiss) at 4646x magnification. ####
 Synaptopodin co-Immunoprecipitation (SP-coIP), RNA-Seq analysis and
 negative contrast electron microscopy Six tissue cultures per biological
 replicate were washed in coIP-solution and dounce homogenized on ice in a
 solution containing (150 µl per sample, coIP-solution, in mM): 10 HEPES,
 120 NaCl, 0.05% (v/v) NP-40 or IGEPAL CA-630 (Sigma Aldrich, #I8896),
 cOmplete protease inhibitor (Roche, according to manufacturer’s
 instructions), murine RNase inhibitor (1:1000, New England Biolabs,
 #M0314) and cycloheximide (0.5 mg/ml, Sigma Aldrich, #C-4859). Input was
 precleared twice with protein A beads (10 µl per 100 µl
 sample/supernatant) to reduce non-specific binding. Protein A beads (10
 µl, New England Biolabs, #S1425) were washed twice in coIP-solution and
 consecutively incubated for 1 hour at 4°C with an anti-synaptopodin
 antibody (rabbit anti-synaptopodin, Synaptic Systems, #163002). Beads were
 carefully washed twice to remove unbound antibodies and incubated for 1
 hour with the precleared supernatant (‘input’) at 4 °C with head-over-tail
 rotation. 10 µl of the precleared supernatant were kept as input control.
 After incubation, beads were washed twice with coIP-solution and finally
 dissolved in RNA protection buffer (New England Biolabs). In another round
 of experiments, hippocampal tissue from adult animals was used for
 co-immunoprecipitation experiments (antibody: monoclonal rabbit
 anti-synaptopodin, Abcam, #ab259976 Abcam) with subsequent negative
 contrast electron microscopy. Beads (30 µl) were adsorbed on carbon-coated
 films stabilized by a copper grid and stained with 3% uranyl acetate
 (Serva, Heidelberg, Germany). Samples were immediately examined with a
 transmission electron microscope (Morgagni 268, 80 kV, FEI, Eindhoven, The
 Netherlands). RNA was released from the beads through protein K lysis
 (Monarch® Total RNA Miniprep Kit; New England Biolabs, #T2010S). After
 lysis, beads were removed from the solution in a magnetic rack and the RNA
 containing supernatant was transferred to fresh tubes. RNA isolation on
 coIP- and input-samples was then performed according to the manufacturer’s
 instructions. RNA content was determined using the Agilent RNA 6000 Pico
 Kit (Agilent, #5067-1513) with a 2100 Bioanalyzer (Agilent, #G2939BA). RNA
 Library preparations for Synpo-related transcriptome analysis were
 performed using the NEBNext® Single Cell/Low Input RNA Library Prep Kit
 for Illumina® (New England Biolabs, #E6420) according to the
 manufacturer's instructions. 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) having at least a phred quality of 30 (&gt; 90
 % of total reads). #### Regional mRNA library preparations and
 transcriptome analysis RNA Library preparations for transcriptome analysis
 were performed using the NEBNext® Single Cell/Low Input RNA Library Prep
 Kit for Illumina® (New England Biolabs, #E6420) according to the
 manufacturer's instructions. Briefly, isolation of the dentate gyrus
 and CA3 area from individual tissue cultures was performed using a
 scalpel. One isolated dentate gyrus or CA3 area respectively was
 transferred to 7.5 µl lysis buffer (supplemented with murine RNase
 inhibitor) and homogenized using a pestle. Samples were centrifuged for 30
 s at 10,000 g 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 with 12 PCR
 cycles. cDNA yield was consecutively analyzed by a High Sensitivity DNA
 assay on a Bioanalyzer instrument (Agilent). cDNA amount 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 was 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 100 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) having at least a phred quality of 30 (&gt;90% of total reads). ####
 Experimental Design and Statistical Analysis Electrophysiological data
 were analyzed using pClamp 10.7 (Axon Instruments) and MiniAnalysis
 (Synaptosoft) software. The fraction of action potentials followed by
 time-locked excitatory postsynaptic current responses was considered as
 synaptic response rate. uEPSC amplitude was assessed in uEPSCs from
 successfully transmitted action potentials as well as the mean amplitude
 of all successfully evoked postsynaptic responses. In order to ensure
 analysis of time-locked unitary responses, events were included if &gt; 5%
 of presynaptic pulses were successfully transmitted for each pulse
 respectively. For individual pulses in pulse trains that caused ≤ 5%
 successfully transmitted events, response rate was set to ‘0’. sEPSC
 properties were analyzed using the automated template search tool for
 event detection. Since sEPSC kinetics might contain information on their
 synaptic origin (in CA3-PCs, large EPSCs have been identified to originate
 from hippocampal mossy fibers), we performed a hierarchical analysis of
 sEPSCs based on their amplitude. All events of a recorded cell were sorted
 based on their peak amplitude. Events above the 50th percentile therefore
 correspond to all events above the cells’ median amplitude of events.
 Amplitude and half width data from sEPSC analysis have been plotted to
 visualize sEPSC kinetics and linear fitting was employed to detect changes
 in parameter interdependencies. sEPSC area was reported as a function of
 amplitude and half width. Input resistance was calculated for the
 injection of –100 pA current at a time frame of 200 ms with a maximum
 distance to the initial hyperpolarization. Resting membrane potential was
 calculated as the mean baseline value. AP detection was performed using
 the input-output curve threshold search event detection tool, and the AP
 frequency was assessed by the number of APs detected during the respective
 current injection time. 3D reconstructions of individual dendritic spines
 in electron micrograph image stacks were performed using the TrakEM2 tool
 in the Fiji software environment by 3 individual investigators blind to
 experimental conditions and hypothesis. From one image stack, subfields
 were selected (proximal to soma: dentate gyrus (DG), 10-50 µm; CA3, 30-90
 µm distance to soma; distal to soma: &gt;100 µm distance to soma in both
 subfields) and all pre- and postsynaptic compartments of asymmetric
 synapses that were fully captured on image stacks were manually
 reconstructed. In order to analyze synaptic complexity, the images with
 the 3D-reconstructed synaptic compartments were used. The number of
 postsynaptic compartments and their postsynaptic densities were manually
 counted for each respective presynaptic compartment by an investigator
 blinded to experimental conditions. Transmission electron microscopy
 images of mossy fiber synapses were saved as TIF-files and analyzed using
 the ImageSP Viewer software ().
 Ultrastructural features were analyzed by an investigator blind to
 experimental conditions in randomly selected mossy fiber terminals from
 electron micrographs of the stratum lucidum in non-lesioned and lesioned
 wildtype tissue cultures. 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 sequencing, low quality, and short
 reads were removed via the CUTADAPT tool. Reads were mapped using RNA STAR
 with the mm10 full reference genome (Mus musculus). 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 analysis was performed from RNA STAR output. Only
 samples that contained &gt;60% uniquely mapping reads (feature: “exon”)
 were considered for further analysis. Statistical evaluation was performed
 using DESeq2 with “pathway integrity” (ECL experiments) or “coIP”
 (synaptopodin-coIP experiments) as the primary factor that might affect
 gene expression. Genes with a low number of mean reads were excluded from
 further analysis. Genes were considered as differentially expressed or
 enriched if the adjusted p-value was &lt; 0.05. Data visualization was
 performed according to a modified version of a previously published
 workflow. Further functional enrichment analyses were 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
 100-500 terms were considered for illustration. Heatmaps were generated
 based on z-scores of the normalized count table. For transcriptome
 analysis following synaptopodin-coIP, DESeq2 analysis was performed to
 compare coIP and input samples. Differentially enriched genes in coIP
 (padj &lt; 0.05 and log2(FC) &gt; 0) were considered for further analyses.
 To correct for non-specific antibody binding, DESeq2 output (log2(FC))
 from the same experimental procedure in synaptopodin-deficient tissue
 cultures was subtracted from log2(FC) of differentially enriched genes in
 wildtype cultures. Only those differentially expressed genes that showed
 &gt; 30% enrichment in FC after Synpo-KO correction were considered as
 significantly enriched. Data were statistically analyzed using GraphPad
 Prism 7 or 9 (GraphPad software, USA). For statistical comparison of two
 experimental groups, a Mann-Whitney test was employed. For statistical
 comparison of three experimental groups, a Kruskal-Wallis test was
 employed. In the graphs demonstrating volumes of the synaptic
 compartments, the box depicts 25-75 percentile, whiskers depict 10-90
 percentile and the line indicates the median. Values outside this range
 were indicated by individual dots. Otherwise, values represent the mean ±
 standard error of the mean (s.e.m.). sEPSC amplitude/frequency plots and
 AP-frequency plots were statistically assessed by the repeated measure
 (RM) two-way ANOVA test with Sidak’s (two groups) multiple comparisons
 test. uEPSC amplitude values from individual cells were stacked in
 subcolumns and the pulse number was defining tabular rows (COLUMN factor:
 pathway integrity, genetic background; ROW factor: EPSC amplitude bin or
 current injection). P-values &lt; 0.05 were considered statistically
 significant (*p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001); results that
 did not yield significant differences are designated ‘ns’. Statistical
 differences in XY-plots were indicated in the legend of the figure panels
 (*) when detected through multiple comparisons, irrespective of their
 localization and the level of significance. ## Code/Software Data were
 analyzed using GraphPad Prism 7+9 (GraphPad software, USA). The enrichment
 of gene sets were tested using the g:Profiler platform.