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: andreas.vlachos@anat.uni-freiburg.de and lenz.maximilian@mh-hannover.de

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 [> 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 > 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 (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.

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 (> 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 (>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 > 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: >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 >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 < 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 < 0.05 and log2(FC) > 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 > 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 < 0.05 were considered statistically significant (p < 0.05, **p < 0.01, *p < 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.