DOI: 10.5061/dryad.tdz08kq2x
title: The amyloid precursor protein regulates synaptic transmission at medial perforant path synapses

description: The perforant path provides the primary cortical excitatory input to the
 hippocampus. Due to its important role in information processing and
 coding, entorhinal projections to the dentate gyrus have been studied in
 considerable detail. Nevertheless, synaptic transmission between
 individual connected pairs of entorhinal stellate cells and dentate
 granule cells remains to be characterized. Here, we have used mouse
 organotypic entorhino-hippocampal tissue cultures of either sex, in which
 the entorhino-dentate (EC-GC) projection is present and EC-GC pairs can be
 studied using whole-cell patch clamp recordings. By using cultures of
 wildtype mice, the properties of EC-GC synapses formed by afferents from
 the lateral and medial entorhinal cortex were compared and differences in
 short-term plasticity were identified. Since the perforant path is
 severely affected in Alzheimer´s disease, we used tissue cultures of
 amyloid-precursor protein (APP)-deficient mice to examine the role of APP
 at this synapse. APP deficiency altered excitatory neurotransmission at
 medial perforant path synapses, which was accompanied by transcriptomic
 and ultrastructural changes. Moreover, presynaptic but not postsynaptic
 APP deletion through the local injection of Cre-expressing
 adeno-associated viruses in conditional APPflox/flox tissue cultures
 increased the neurotransmission efficacy at perforant path synapses. In
 summary, these data suggest a physiological role for presynaptic APP at
 medial perforant path synapses that may be adversely affected under
 altered APP processing conditions.
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
 Goethe-University Frankfurt, Faculty of Medicine, and the
 Albert-Ludwigs-University Freiburg, Faculty of Medicine.
 <strong>Preparation of organotypic tissue
 cultures </strong> Entorhino-hippocampal tissue cultures were
 prepared at postnatal day 4 - 5 from C57BL/6J<em> </em>and
 APP-deficient (APP<sup>-/-</sup>; null allele; (Li et al.,
 1996; Heber et al., 2000)) animals of either sex as previously described
 (Del Turco and Deller, 2007). Briefly, the dorsal brain surface was
 immobilized on cutting plates with Histoacryl<sup>®</sup> (B.
 Braun) and transverse sectioning was performed using a vibratome (Leica,
 VT1200S). The entorhino-hippocampal complex was dissected from 300 µm
 transverse sections containing the entorhinal cortex and ventral
 hippocampus (c.f. Figure 1) and placed on porous filter membranes
 (Millipore, PICM0RG50). Compared to hippocampus-only tissue cultures,
 mossy cell axons are limited to the inner molecular layer of the dentate
 gyrus and ectopic mossy fiber sprouting cannot be observed (Becker et al.,
 2012). Conditional APP-deficient cultures were prepared from homozygous
 APP<sup>flox/flox</sup> animals (Mallm et al., 2010) of either
 sex that were crossed to
 B6.Cg-Gt(ROSA)26Sor<sup>tm14(CAG-tdTomato)Hze</sup>/J
 (Ai14<sup>+/-</sup>; Jackson Laboratories #007914; (Madisen et
 al., 2010)). The newly generated mouse line
 (Ai14-APP<sup>flox/flox</sup>) is a reporter mouse revealing
 nuclear recombination by cellular tdTomato expression. Here, both the
 promoter and exon 1 of the APP gene were flanked by loxP sites, which
 results in cell-type specific null alleles upon Cre-recombination (Mallm
 et al., 2010). 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 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 (Hailer et al., 1996; Vlachos et al., 2012a;
 Vlachos et al., 2013a; Humpel, 2015). Experiments were carried out in
 dentate granule cells from the suprapyramidal blade. Due to the
 three-dimensional orientation of the hippocampus, the cultured dentate
 gyrus may show morphological variations of the infrapyramidal blade in
 some tissue cultures. <strong>Perforant path tracing and local viral Cre-GFP
 expression</strong> Adeno-associated viruses (AAV) obtained from
 SignaGen Laboratories, Maryland (AAV2-Synapsin-tdTOMATO, #SL100896 and
 AAV2-Synapsin-GFP, #SL100817; both 10<sup>13</sup> vg/ml, 1:4
 diluted in PBS) and the University of North Carolina Vector Core (UNC
 Vector Core; AAV2-hSyn-GFP-Cre; 10<sup>12</sup> vg/ml, 1:4
 diluted in PBS) were injected into the entorhinal cortex at 3–5
 days<em> in vitro</em> at a Zeiss Axioscope 2 equipped with a
 4x objective (air, NA 0.1) using borosilicate glass pipettes (c.f. (Lenz
 et al., 2019)). 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. <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. To label calretinin, whole tissue cultures were incubated with rabbit anti calretinin (1:1000; Synaptic Systems, #214102) 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). TO-PRO<sup>®</sup>or DAPI (1:5000 in PBS for 10 min; TO-PRO<sup>®</sup>: Invitrogen, #T-3605; DAPI: Thermo Scientific, #62248) nuclear stain 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 60x objective lens (NA 1.4, Leica). <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 488- or Alexa 647-conjugated streptavidin (1:1000 in PBS with 10 % NGS, 0.1 % Triton X-100; Invitrogen, #S-32354 and #S-32357 respectively) for 4 h and DAPI or TO-PRO<sup>®</sup> staining was used to visualize cytoarchitecture (1:5000 in PBS for 10 min; TO-PRO<sup>®</sup>: Invitrogen, #T-3605; DAPI: 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 Nikon Eclipse C1si laser-scanning microscope with a 4x objective lens (NA 0.2, Nikon), a 20x objective lens (NA 0.9, Nikon) and a 60x objective lens (NA 1.4, Nikon) or a Leica SP8 confocal microscope equipped with a 40x objective lens (NA 1.3, Leica). Detector gain and amplifier were initially set to obtain pixel intensities within a linear range. <strong>Transmission Electron Microscopy</strong> APP<sup>+/+</sup> and APP<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 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 in 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 4646x magnification. Acquired images were saved as TIF files and analyzed using the ImageSP Viewer software (http://e.informer.com/sys-prog.com). In each group, 50 synapses from 5 independent tissue cultures were analyzed in the distal parts of the molecular layer. Asymmetric spine synapses were identified and the total amount of presynaptic vesicles and docked vesicles to presynaptic active zones were manually quantified by an investigator blind to the genotype.  <strong>Paired whole-cell patch-clamp recordings </strong> Whole-cell voltage-clamp recordings from dentate granule cells 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. In some experiments, Alexa488 was added to the internal solution to visualize neuronal morphology prior to recordings. Cells were visually identified using an LN-Scope (Luigs and Neumann, Ratingen, Germany) or a Zeiss LSM750 equipped with infrared dot-contrast and a 40× water-immersion objective (numerical aperture [NA] 0.8; Olympus). Granule cells were patched in the outer 1/3 of the granule cell layer. Electrophysiological signals were amplified using a Multiclamp 700B amplifier, digitized with a Digidata 1550B digitizer, and visualized with the pClamp 11 software package. Neurons were recorded at a holding potential of -70 mV. To avoid the recording of immature granule cells, recordings were discarded if the initial resting membrane potential was higher than -70 mV. Hilar mossy cells were identified during the experiment by their morphological and electrophysiological features (Scharfman and Schwartzkroin, 1988; Henze and Buzsaki, 2007): large multipolar cell body, sag-current upon hyperpolarization, small or absent after-hyperpolarization upon action potential induction, resting membrane potential &gt; -70 mV, and synaptic connection (depolarizing) to postsynaptic dentate granule cell.  Recordings were discarded if series resistance reached ≥ 30 MΩ.  Action potentials were generated in the presynaptic cell by 5 ms square current pulses (1 nA) elicited at 0.2 Hz (up to 50 pulses), while recording unitary excitatory postsynaptic currents (uEPSCs) from dentate granule cells. Neurons were considered to be connected if &gt; 5 % of action potentials evoked time-locked inward uEPSCs. For short-term plasticity, 5 action potentials were applied at 10 or 20 Hz, respectively (inter-sweep-interval: 5 sec, up to 30 repetitions). <strong>Regional mRNA library preparation and transcriptome analysis</strong> RNA library preparations for transcriptome analysis were performed using the NEBNext® 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 from individual tissue cultures was performed using a scalpel. One isolated dentate gyrus was transferred to 7.5 µl lysis buffer (supplemented with murine RNase inhibitor) and homogenized using a pestle. 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 with 12 PCR cycles. 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 with 0.8X SPRI beads (Beckman Coulter, #B23318) 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; (Galaxy, 2022)). All files contained more than 10 M high-quality reads (after mapping to reference genome; mm10) with a phred quality of at least 30 (&gt;90% of total reads). <strong>Compartmental modeling</strong> Compartmental simulations of synaptic EPSCs in dentate granule cells were performed as described before (Vlachos et al., 2012c) using the simulation environment T2N (Trees-to-NEURON interface, (Beining et al., 2017)) linking Matlab (Mathworks, version 2018b) and NEURON (version 7.4; (Hines and Carnevale, 1997); www.neuron.yale.edu). Briefly, we used 8 reconstructed morphologies of mouse dentate granule cells (Schmidt-Hieber et al., 2007) from ModelDB (accession number 95960). Passive biophysical properties were adjusted to match the intrinsic properties of wildtype granule cells in organotypic tissue cultures from Galanis and colleagues (Galanis et al., 2021). Excitatory (AMPA-like) synaptic currents were simulated using a double-exponential conductance change with a peak amplitude of 0.06 nS, a rise time of 0.2 ms, a decay time of 2.5 ms, and a reversal potential of 0 mV. To determine the dependence of simulated EPSC amplitudes on the distance from the soma, identical single synaptic input was activated at different locations along a path between the soma and a distal end of the dendrite, and corresponding EPSCs were detected at the soma. Simulated cells were voltage clamped at −70 mV. To simulate compound EPSCs, we monitored voltage-clamped somatic currents in 8 model granule cells in which synaptic activity was triggered by sequential activation of a single dendritic AMPA synapse placed at decreasing distances in one dendritic branch or by synchronous activation of dendritic AMPA synapses placed at equidistant locations in all dendritic branches. <strong>Experimental Design and Statistical Analysis</strong> STUDY DESIGN In this study, we used age-matched organotypic entorhino-hippocampal tissue cultures in a prospective study design. Each tissue culture represents a biological replicate that defines independent experiments. Excitatory inputs onto dentate granule cells were analyzed in age-matched tissue cultures, in which one pathway was tested per tissue culture with 1-5 postsynaptic cells. Only one tissue culture per mouse was used for the investigation of a distinct pathway. Each pair is considered a biological replicate. To elucidate the role of APP in synaptic transmission, age-matched APP-deficient and C57BL/6J cultures were compared (Figures 4, 5). In the localized APP-recombination experiments (Figure 6), tissue cultures from APP<sup>flox/flox</sup> x Ai14 Cre reporter mice were used and recombination was achieved by local viral injection of AAV-hSyn-Cre-GFP. For postsynaptic recombination, viral injections were performed in the dentate gyrus. For presynaptic recombination, viral injections were performed in the medial part of the entorhinal cortex. Recombination was determined during the patch-clamp experiments by visual inspection of the tdTomato signal. QUANTIFICATION Electrophysiological data were analyzed using pClamp 10.7 (Axon Instruments) and MiniAnalysis (Synaptosoft) software. The fraction of action potentials not followed by time-locked excitatory postsynaptic current responses was considered as synaptic failure rate. The uEPSC amplitude, rise time (rise50), and area were assessed in uEPSCs from successfully transmitted action potentials, as well as the mean amplitude of all successfully evoked postsynaptic responses. For evaluation of short-term plasticity experiments, recorded traces were averaged in Clampfit 10.7, irrespective of successful synaptic transmission at individual pulses. In some analyses, postsynaptic responses were normalized to the first response in averaged traces. Presynaptic ultrastructural features were analyzed in randomly selected perforant path terminals from electron micrographs of the outer parts of the molecular layer. Postsynaptic features of the same set of images were analyzed in a previous study (Galanis et al., 2021). Presynaptic terminals and vesicles were manually assessed by an investigator blind to experimental conditions. RNA sequencing data were uploaded to the galaxy web platform (public server: usegalaxy.eu; Afgan et al., 2018; Jalili et al., 2020; Afgan et al., 2016) and transcriptome analysis was performed using the Galaxy platform in accordance with the reference-based RNA-seq data analysis tutorial (Batut et al., 2021). 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 (&lt; 50 counts) were excluded from further analysis.  Read counts were further processed with R (v 4.2.1). Differential gene expression analysis was performed with DESeq2 R package (v 1.36; (Love et al., 2014)) using Wald significance test. Gene-set enrichment analysis was performed with the clusterProfiler R package (v 4.4.4; (Wu et al., 2021)) using gene-sets from MsigDB (v 7.2; (Subramanian et al., 2005)). In both analyses, the adjusted p-value below 0.05 was set as significant threshold. STATISTICAL ANALYSIS Data were statistically analyzed using GraphPad Prism 9 (GraphPad software, USA). For statistical comparison of two experimental groups, a Mann-Whitney test was employed. For the evaluation of data sets with three experimental groups, a Kruskal-Wallis test followed by Dunn’s posthoc correction was performed. Short-term plasticity experiments were statistically assessed by the repeated measure (RM) two-way ANOVA test with Sidak’s multiple comparisons test. uEPSC amplitude values from individual cells were stacked in subcolumns and pulse number defined tabular rows (COLUMN factor: pathway, genetic background or recombination; ROW factor: pulse number). 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 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.) unless otherwise indicated. <strong>Data and materials availability</strong> Source data with statistical evaluations are provided here. Raw data (.fastq-files) used for transcriptome analysis are available at the Gene Expression Omnibus; accession number: GSE213284. The code for compartmental modeling will be available via the Zenodo repository. 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).