Animals

Adult C57BL/6 J mice (Prnp+/+) were purchased from Charles River Laboratories (Paris, France). PrnpZH3/ZH3 mice line was provided by A. Aguzzi (Switzerland) (see [50] for details). PrnpZH1/ZH1 mice [24] were purchased from the European Mouse Mutant Archive (EMMA, Monterotondo, Italy). A total of 185 adult (3–5 months old) male mice (ZH3: Prnp+/+ = 81 and PrnpZH3/ZH3 = 84; ZH1: Prnp+/+ = 10 and PrnpZH1/ZH1 = 10) were used in the present study. In ZH1 mouse experiments, null PrnpZH1/ZH1 and control mice (Prnp+/+) were obtained by crossing heterozygous Prnp+/ZH1 mice to obtain a mixed background (B6.129). It is well described that behavior and neural physiology are different between male and female rodent models due to several hormone and non-hormone-derived reasons [88]. Thus, we used only males in order to establish an equivalent group comparable with previous publications. All experiments were performed following the protocols and guidelines of the Ethical Committee for Animal Experimentation (CEEA) of the University of Barcelona. CEEA of the University of Barcelona approved the protocol for using animals in this study (CEEA approval #276/16 and 141/15). Behavioral and electrophysiological studies were performed following the guidelines of the European Union Council (2010/276:33-79/EU) and current Spanish regulations (BOE 34:11370-421, 2013) for the use of laboratory animals in chronic experiments. Experiments were also approved by the Ethics Committee for Animal Care and Handling of the Pablo de Olavide University (UPO-JA 06/03/2018/025).

Immunoblotting

Proteins from brain tissue lysates or primary cortical neurons were extracted using RIPA buffer with protease and phosphatase inhibitor cocktails (Roche). Total lysates were obtained by 30 s of centrifugation at 4 °C. The protein concentration of the lysates was quantified using Pierce BCA Protein Assay Kit (Thermo Scientific). Then, 10–50 μg of proteins were loaded to SDS-PAGE gels and transferred and transferred to nitrocellulose membranes for 1 h. Membranes were blocked with Tris-buffered solution with 0.1% tween, 5% skimmed milk, and 2% of FBS for 1 h at room temperature (RT) and incubated with PSD95 (1:1.000, MAB1598; Millipore), PrP (1:500; 6H4; Thermo) or Actin (1:20.000; MAB1501; Millipore) antibodies at 4 °C O/N. Following HRP-linked secondary antibody (Dako) incubation for 1 h at RT, membranes were developed with ECL substrate (Thermo).

Behavioral studies

A total of 147 animals were used in these sets of experiments (ZH3: Prnp+/+ = 63 and PrnpZH3/ZH3 = 64; ZH1: Prnp+/+ = 10 and PrnpZH1/ ZH1 = 10). Mice were housed alone in boxes on a 12/12 h light/dark cycle with constant ambient temperature (21 ± 1 °C). Water and food were provided ad libitum except for the instrumental learning tests (see below).

Nest building

For this test, a total of 14 mice (3 months old) were used (PrnpZH3/ZH3 = 7 and Prnp+/+ = 7). On the first day of testing, one piece of tissue paper (36 × 12 cm) was placed in the cage to facilitate nest building (Additional file 1: Fig. S1). The presence and the quality of each nest were photo-documented and evaluated the following day according to a modified 5-point scale using the method described by Deacon [89]. Two different blinded researchers evaluated the nest generated by each mouse. Data are presented as the mean ± S.E.M. in (Additional file 1: Fig. S1). The statistical analysis was performed with the Mann-Whitney non-parametric test (GraphPad Prism 8 software).

Open field test

In this test, PrnpZH1/ZH1 mice were not used since detailed studies were already developed using this model [45, 54]. In our experiments, mice (Prnp+/+ = 49 and PrnpZH3/ZH3 = 49) were placed in a square open field altimeter box (35 × 35 × 25 cm, Cibertec, Madrid, Spain). The field had a grid (16 × 16 cm) of infrared lasers on the XY-axis and one on the Z-axis. Locomotor activity was measured for 15 min in mice with the MUX-XYZ16L software. Mice were placed in the box’s periphery for 15 min for two consecutive days, and their behavior was recorded. The first day was considered a training session to reduce mouse anxiety associated with manual handling, and the data analyzed and displayed in the manuscript corresponded to the second session. The system inferred mouse activity by counting laser intersections. For anxiety-related behavior measurement, the center (inner) square of the field (10 × 10 cm) was considered as the central zone and the rest of the square as the peripheral (outer) zone [56] (see Fig. 1a). For quantification and to distinguish motility from exploratory behavior, it was considered that a mouse spent time in one of the regions (center vs. periphery) if it remained in the region at least 3 s. Rearing episodes were considered when the animal stood up for at least 3 s, and immobility episodes if immobile for an additional 3 s. Obtained data were analyzed, and the sum of the crossed X- and the Y-axes are presented together to show total mouse mobility in the experiments. The time spent in the maze periphery zones measures thigmotaxis or wall-hugging behavior and indicates anxiety-related behavior [56]. Data are presented as the mean ± S.E.M. The statistical analysis was performed with a T-test or Mann-Whitney U non-parametric test (GraphPad Prism 8 software). The asterisks indicate significant differences: **p < 0.01 and ***p < 0.001. The arena and the walls were cleaned with soap and ethanol between trials to remove olfactory cues between experiments.

Operant conditioning tests

The instrumental learning tests were performed as described in previous studies of our group [90]. Six Skinner boxes were used simultaneously (12.5 × 13.5 × 18.5 cm; MED Associates, St. Albans, VT, USA). Each Skinner box was housed in a sound-attenuating cubicle (90 × 55 × 60 cm) constantly exposed to white noise (± 45 dB) and dim light (Cibertec, S.A, Madrid, Spain). The boxes had a trough to receive food pellets (Noyes formula P; 45 mg; Sandown Scientific, Hampton, UK) by pressing a lever. Before the test, mouse food availability was monitored for 7 days to reduce initial mouse weight to 85%. First, mice (ZH3: Prnp+/+ = 49 and PrnpZH3/ZH3 = 49; and ZH1: Prnp+/+ = 10 and PrnpZH1/ZH1 = 10) were trained to press the lever to receive food pellets in a fixed-ratio (1:1) schedule. Seven daily sessions (20 min/each) were held. The boxes were cleaned with soap and ethanol (30%) between trials. Obtaining ≥ 20 pellets for two consecutive sessions was defined as the criterion to assume the learning criteria achievement. Following this first operant conditioning test, we increased the paradigm complexity to test the mice in a more demanding cognitive task for an additional 10 days. Only animals that met the learning criterion were tested (ZH3: Prnp+/+ = 24 and PrnpZH3/ZH3 = 20; and ZH1: Prnp+/+ = 8 and PrnpZH1/ZH1 = 5). The paradigm consisted of light (ON period) and dark periods (OFF period) randomly distributed during the session. The light was provided by a small light bulb located over the lever. During the ON period (20 s), lever presses were reinforced with food pellets at a ratio of 1:1. During the OFF period, lever presses were not rewarded and were penalized by adding ten additional seconds (20 ± 10 s) to the next ON period. The number of lever presses during the different conditioning paradigms was monitored and recorded with the MED-PC program (MED Associates, St. Albans, VT, USA). Statistical analysis was carried out using two-way ANOVA with repeated measures and Bonferroni’s multiple comparisons test (GraphPad Prism 8 software). Asterisks indicate significant differences: **p < 0.01; and ***p < 0.001. Data are presented as the mean ± S.E.M. or as a percentage (as indicated in each figure).

Rotarod test

For this test, a total of 15 mice of 4 months were used (PrnpZH3/ZH3 = 8 and Prnp+/+ = 7). Motor performance was tested using an accelerating rotarod. Mice were pre-trained to the task to reach a minimum of 30 s performance at 5 rpms on the 1st day of testing. In each training run, animals were placed on the rods at an initial speed of 5 rpm for 30 s. After that, the testing consisted of 5 consecutive trials with 15-min inter-trial intervals. Each trial consisted of 30 s at 5 rpm followed by 5 rpm increases every 15 s with a cut-off of 5 min. Results are expressed as the mean latency of animals to fall from the rod ± S.E.M. The statistical analysis was performed with the two-way ANOVA + Bonferroni’s multiple comparisons test (GraphPad Prism 8 software).

Object recognition test

The object recognition test was performed in a homemade arena (30 × 25 × 20 cm), as described [91]. A total of 23 Prnp+/+ and 24 PrnpZH3/ZH3 mice were analyzed. Additionally, 6 PrnpZH1/ZH1 mice were also used with 7 Prnp+/+ counterparts. The test consisted of four phases of 10 min/each. First, animals were habituated to the field without any object (habituation session). One hour later, two identical plastic objects were placed in the center of the arena for the training session. A short-term memory test was performed 2–3 h later by changing one of the objects (see Additional File 4: Fig. S4). ZH1 mouse mobility was expressed as the number of rearing episodes during the habituation session. The arena and the objects were cleaned with soap and 30% ethanol between trials to remove olfactory cues. Mouse behavior was recorded with a video camera placed over the arena, and these recordings were used to measure the exploratory behavior blindly. Sniffing and gently touching the objects were counted as exploratory behavior. To further support increased anxiety levels in the PrnpZH3/ZH3 mice, fecal bodies left in the maze during the habituation session were counted by the observer once the test subject was removed since it has been demonstrated that highly emotional animals exhibit increased defecation [56]. Statistical analysis was performed with the Mann-Whitney U non-parametric test (GraphPad Prism 8 software). Data are presented as the mean ± S.E.M. or as a percentage (indicated in each figure).

Mouse surgery

A total of 98 adult male (3-5 months) mice were implanted with stimulating and recording electrodes (Prnp+/+ = 49 and PrnpZH3/ZH3 = 49). Four of them died during surgery, and 33 mice were excluded because of the inability to obtain reliable and clean recordings. Thus, the experiments were performed with 61 mice (Prnp+/+ = 31 and PrnpZH3/ZH3 = 30). Surgery was performed as described in [19, 92]. Mice were deeply anesthetized with ketamine (35 mg/kg) and xylazine (2 mg/kg), and electrodes were aimed at the right dorsal hippocampus. Two recording electrodes were implanted in the stratum radiatum of the CA1 area (2.2 mm caudal to Bregma, 1.2 mm lateral, and 1.3 mm ventral), and two stimulating electrodes were implanted in the Schaffer collateral pathway of the CA3 region (1.5 mm posterior to Bregma, 2 mm lateral, and 1.3 mm ventral). Electrodes were made of 50 μm Teflon-coated tungsten wires (Advent Research, Eynsham, UK). Electrode localizations were checked according to the field excitatory postsynaptic potential (fEPSP) profile evoked by a single stimulation. A silver wire was fixed to the skull as ground. All the wires were soldered to a six-pin socket (RS Amidata, Madrid, Spain) fixed to the skull with dental cement. Recordings were started at a minimum of 1 week after the surgery.

Electrophysiology recordings

Animals were consecutively recorded in groups of six individuals since they reach the total number of animals used in each experiment. Each animal was placed in a small plastic cubicle (5 × 5 × 10 cm) inside a large Faraday box (30 × 30 × 20 cm). fEPSPs were recorded with a high impedance probe (2 × 1012 Ω, 10 pF) using differential amplification at a bandwidth of 0.1 Hz–10 kHz (P511, Grass-Telefactor, West Warwick, RI, USA). For each experiment, artefactual recordings were discarded. The stimulation intensity threshold of each animal was set with paired-pulse stimulations at 40 ms of inter-stimulus interval. The stimulus intensity was set to 40–60% of the amount necessary to evoke a suturing response. These intensity values were used for all the experiments.

Paired-pulse stimulation

For synaptic facilitation experiments, 51 mice (Prnp+/+ = 27 and PrnpZH3/ZH3 = 24) were stimulated at Schaffer collaterals with a pair of pulses at different inter-stimulus intervals (10, 20, 40, 100, 200, and 500 ms) at 2 × threshold intensities (≈ 0.2 mA). Threshold values were previously defined for each mouse. As classically defined, threshold values were determined as the intensity evoking fEPSP responses in 50% of the cases. For all the inter-pulse intervals, the stimulations were repeated ten times. Data are represented as the mean percentage increases of fEPSP2 from fEPSP1 recordings (fEPSP2 / fEPSP1 × 100) ± S.E.M.

Input/output curves

Schaffer collaterals of 29 mice (Prnp+/+ = 14 and PrnpZH3/ZH3 = 15) were stimulated with paired pulses at 20 increasing intensities (from 0.02 to 0.4 mA, increased in steps of 0.02 mA) at 40 ms of inter-stimulus interval. For all the selected intensities, the stimulations were repeated ten times. Data are represented as the mean of fEPSP slopes (V/s) ± S.E.M. The same data are presented as the mean of paired-pulsed ratio (PP ratio) ± S.E.M. PP ratio is the percentage of the increase of the fEPSP2 from fEPSP1 recordings (fEPSP2 / fEPSP1 × 100). The area under the curve (AUC) was calculated from the PP ratio of all the animals using GraphPad Prism 8 software. Statistical analysis was performed using the Mann-Whitney-Wilcoxon non-parametric test or two-way ANOVA + Bonferroni’s multiple comparisons test (GraphPad Prism 8 software). The asterisks indicate significant differences: *p < 0.05; **p < 0.01; and ***p < 0.001 in the figure.

Long-term potentiation experiments

For long-term potentiation experiments, 40 mice (Prnp+/+ = 20 and PrnpZH3/ZH3 = 20) were stimulated at Schaffer collaterals. In a first experimental step, fEPSP baseline values were evoked and recorded for 15 min, with paired-pulse stimulus presented every 20 s (40 ms inter-stimulus). Stimulus intensities were selected to evoke fEPSPs of about 0.2–0.3 mV of amplitude (see insets in Fig. 4a,b). Next, LTP was evoked with a high-frequency stimulation (HFS) protocol. HFS consisted of five trains of pulses at a rate of 1/s (200 Hz, 100 ms) with the same intensity as the baseline recording. The HFS was repeated six times at intervals of 1 min. After the HFS protocol, fEPSPs were recorded, as for baseline, for 1 h. The following 4 days, the recordings were repeated for 30 min. fEPSPs and 1 V rectangular pulses corresponding to stimulus presentations were saved on a PC using an analog/digital converter (CED 1401 Plus, Cambridge, England). Data were analyzed offline using Spike2 and Signal 5.04 software with homemade representation programs [58]. Collected recordings were represented and analysed offline with the help of commercial (Spike 2 and Signal 5.04) programs following procedures described elsewhere. The slope of collected fEPSPs was computed as its first derivative (volts/s). Five successive fEPSs were averaged and the mean value of the slope was determined. Data are presented as the mean of the percentage compared to the baseline ± S.E.M. The statistical analysis was performed using two-way ANOVA + Bonferroni’s multiple comparisons test (GraphPad Prism 8 software). The asterisks and symbols indicate significant differences: *p < 0.05; **p < 0.01; and ***p < 0.001; ##p < 0.01; and ###p < 0.001.

KA-induced epilepsy and seizure analysis

Adult (3–4 months old) male mice were used for these sets of experiments (Prnp+/+ = 18 and PrnpZH3/ZH3 = 20) essentially as described in [28]. A KA (Sigma-Aldrich, Darmstadt, Germany) solution was freshly prepared for each experiment in 0.1 M phosphate buffer. Mice were injected with KA (10 mg/kg b.w.) three times: at 0 min, 30 min, and 60 min. After the first injection, mice were placed in clean boxes (1–3 mice/box). The presence of epileptic seizures was monitored in situ and recorded with a video camera for 3 h after drug administration. Seizure severity was scored in grades following the following criteria: grade I-II: hypoactivity and immobility; grade III-IV: hyperactivity and scratching; grade V: loss of balance control and intermittent convulsions; grade VI: continuous seizures and bouncing activity (also reported as blinking episodes or “pop-corn” behavior). Data are presented as the mean ± S.E.M. or as a percentage (as indicated in each figure). Statistical analysis was performed with the Mann-Whitney U non-parametric test (GraphPad Prism 8 software). The asterisk indicates significant differences: *p < 0.05 in the figure.

RNAseq

Hippocampi were extracted, flash frozen on dry ice, and RNA was harvested using RNAEasy Mini kit (Qiagen). Libraries were prepared using the TruSeq Stranded mRNA Sample Prep Kit v2 according to the manufacturer’s protocol. Briefly, 500 ng of total RNA was used for poly(A)-mRNA selection using Oligo (dT) magnetic beads and subsequently fragmented to approximately 300 bp. cDNA was synthesized using reverse transcriptase (SuperScript II, Invitrogen) and random primers. The second strand of the cDNA incorporated dUTP in place of dTTP. Double-stranded DNA was further used for library preparation. dsDNA was subjected to A-tailing and ligation of the barcoded Truseq adapters. All purification steps were performed using AMPure XP Beads. Library amplification was performed with PCR using the primer cocktail supplied in the kit. Final libraries were analyzed using Agilent DNA 1000 chip to estimate the quantity, check the size distribution, and then quantified by qPCR using the KAPA Library Quantification Kit (KapaBiosystems, Merck, Darmstadt, Germany) before amplification Illumina’s cBot. Libraries were sequenced 1 × 50 bp on Illumina’s HiSeq 2500.

The quality of the fast files was checked using the FastQC software (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). An estimation of ribosomal RNA in the raw data was obtained with riboPicker [93]. Reads were aligned with the STAR mapper [94] to release M14 of the Mus musculus Gencode version of the genome (GRMm38/mm10 assembly) (https://www.gencodegenes.org/mouse/release_M14.html). A raw count of reads per gene was also obtained with STAR (-quantMode TranscriptomeSAM GeneCounts option). The R/Bioconductor package DESeq2 [95, 96] was used to assess differential expression between experimental groups (Wald statistical test + false discovery rate correction). Prior to processing the differential expression analysis, genes for which the sum of raw counts across all samples was less than two were discarded. Deregulated genes with a padj < 0.05 were used to disclose relevant pathway alterations in the REACTOME v77 pathway database gene expression. The analysis has been done just with the protein-coding genes. The gene difference was considered biologically relevant if they are upregulated or downregulated with a fold change of > 1.2 or < 0.85, respectively. A pathway was considered relevant if it was related to neuronal and/or cerebral functions, showed significance (padj < 0.05) and contained more than 10 deregulated genes. The sequencing data have been deposited at the Gene Expression Omnibus (GEO) with accession code: GSE189691 (Matamoros-Angles, A; Hervera, A; Soriano, J; Martí, E; Carulla, P, Llorens, F; Nuvolone, M; Aguzzi, A; Ferrer I; Gruart, A; Delgado-García, JM; Del Río, JA. RNA sequencing of hippocampus of Prnp+/+ and PrnpZH3/ZH3 animals. https://identifiers.org/geo:GSE189691).

RT-qPCR

For RT-qPCR validations, cDNA was obtained with a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems) following the supplier’s instructions. RT-qPCR reactions contained 4.5 μL cDNA and mixed with 0.5 μL 20X TaqMan Gene Expression Assays and 5 μL of 2X TaqMan Universal PCR Master Mix (Applied Biosystems) for a final volume of 10 μL. TaqMan probes used were as follows: Grin2b Mm00433820_m1, Gabrr2 Mm00433507_m1, Kacnj6 Mm01215650_m1, Kcna1 Mm00439977_s1, Kcnj2 Mm00434616_m1, Kcnq3 Mm00548884_m1 (Applied Biosystems). Actb Mm02619580_g1 and Aars Mm00507627_m1 were used as endogenous controls. The assay was performed using technical duplicates per sample in 384-well optical plates with ABI Prism 7900 Sequence Detection system (Applied Biosystems, Life Technologies) following the supplier’s parameters: 50 °C for 2 min, 95 °C for 10 min, and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The Sequence Detection Software (SDS version 2.2.2, Applied Biosystems) was used for data processing. It was further analyzed with the ∆∆Ct method, which consists of obtaining ∆Ct by normalizing each target gene to its endogenous control, followed by subtracting the mean-∆Ct of the control group samples to each ∆Ct to obtain ∆∆Ct values, and finally using these ∆∆Ct values as the negative exponent with base 2, thereby obtaining fold change per sample.

Fluoro-Jade B staining

Mice were perfused 7 days after the KA administration with 4% paraformaldehyde (PFA) dissolved in 0.1 M phosphate buffer (pH = 7.3–7.4). Brains were dissected and postfixed overnight with the same fixative solution. The following day, they were cryoprotected in 0.1 M phosphate buffer containing 30% sucrose (w/v). After freezing in dry ice, 50-μm-thick coronal sections were obtained with a freezing microtome (Leica, Wetzlar, Germany). Sections containing the dorsal hippocampus were selected and rinsed in 0.1 M phosphate buffer and mounted on gelatin-coated slides. The slides containing sections were dried at 37 °C overnight. The following day, they were heated at 50 °C for 45 min before staining to improve adhesion. The staining started with pretreatment for 3 min in absolute alcohol, followed by 1 min in 70% ethanol and 1 min in deionized water. After that, they were oxidized in a solution of 0.06% KMnO4 for 15 min. Following three rinses (2 min/each) in deionized water, they were incubated in a solution of 0.001% Fluoro-Jade B (Chemicon, Temecula, CA, USA) containing 0.05% of DAPI in 0.1% acetic acid for 30 min. Finally, sections were rinsed in deionized water (3 min), dehydrated with ethanol, cleared with xylene, and coverslipped with EukittTM (Merck, Darmstadt, Germany). Considering that wild-type mice did not displayed Fluoro-Jade B labeled cells in the CA1-CA3 regions after KA treatments and only a very few background could be seen at high magnification and long exposures times (> 500 ms), the Fluoro-Jade B fluorescence in the pyramidal layer of the dorsal hippocampal region (4 sections of each mouse, n = 4 mice per genotype) was photo-documented using an Olympus BX61 epifluorescence microscope equipped with a cooled DP12L camera (Hamburg, Germany). Photomicrographs were obtained using a × 40 objective with identical time exposure between preparations from each wild-type and respective knock-out mouse. No modifications were applied to the obtained pictures. Changes in Fluoro-Jade B labeling were determined by analyzing the corrected total cell fluorescence (CTCF) values (see Matamoros-Angles et al. [97], for details) in the pyramidal layer of hippocampal CA1-3 regions of four mice of each genotype, taking a region of interest of 200 × 100 μm centered in the pyramidal layer, and 4–5 different sections per animals were analyzed and averaged. Data were expressed as mean ± S.E.M. The statistical analysis of the obtained data was performed using Mann-Whitney U non-parametric test in GraphPad Prism 8 software. A value of ***p < 0.001 was considered statistically significant in the CTCF analysis.

Primary cortical cultures of Prnp
ZH3/ZH3 and wild-type mice

Primary cortical cultures were fashioned from E16.5–E17.5 Prnp+/+ and PrnpZH3/ZH3 mouse embryos, as explained elsewhere [98]. Brains were removed from the skull and rinsed in cold Hank’s balanced salt solution (HBSS) containing glucose (6.5 mg/ml). The meninges were removed, and the cortical lobes were isolated. Tissue pieces were treated with trypsin for 15 min at 37 °C. After the addition of horse serum followed by centrifugation, cells were isolated mechanically with a polished glass pipette after treatment with 0.025% DNAse for 10 min at 37 °C. One million cells were plated on a 35-mm diameter glass-bottom gridded culture dish (Ibidi, Martinsried, Germany) previously coated with poly-d-lysine (Sigma-Aldrich). NeurobasalTM medium supplemented with 6.5 mg/ml glucose, 2 mM glutamine, penicillin/streptomycin, 5% of horse serum, and B27 was used as a culture medium (all from Invitrogen-Thermo Fisher Scientific, MA, USA). As Prnp0/0-derived cells are sensitive to serum removal [99], after 24 h, the serum was reduced to 2.5%. The medium was changed every 2 days. Horse serum was entirely removed on the eighth day of culture.

Calcium imaging in neuronal culture

Primary cortical neurons were infected 24 h after seeding with AAV9-Synapsin-GCaMP6f [60] (Watertown, MA, USA). In our cultures, the genetically encoded calcium indicators started to express 3–4 days after infection. Calcium changes in GCaMP6f-expressing neurons were recorded at 8, 11, 13, and 15 days in vitro (DIV) using an Olympus IX71 inverted microscope (Olympus, Hamburg, Germany), equipped with an ORCA-Flash 4.0 camera (Hamamatsu Photonics, Japan). During recording, the cells were maintained in a microscope stage incubator at 5% CO2 and 37 °C (Okolab S.R.L., Italy). The same region of the culture was recorded throughout the days following the culture dish grid references. Images (1024 × 1024 pixels) were captured using a × 20 objective and 470 nm wavelength (CoolLED’s pE-300white, Delta Optics, Madrid, Spain) every 100 ms for 8–10 min using the CellSensTM software (Olympus) or the Micro-Manager Open Source Microscopy Software (https://micro-manager.org). Exposure levels and frequency were maintained between cultures and evaluation days. GCaMP6f activity was measured in four different identified squares of each culture dish during these 4 days.

Neuronal activity traces, spike events, and network bursts

The recordings were analyzed offline using two MATLABTM toolboxes: NETCAL (www.itsnetcal.com) [100, 101] and NeuroCa [102]. In NeuroCa, an automatic analysis was performed afterward to corroborate obtained NETCAL results. Using NETCAL, a highly contrasted image of the recording’s average fluorescence was created, and regions of interest (ROIs) were automatically detected as those objects with a circular shape whose brightness was over a preset threshold. NETCAL and NeuroCa software-rendered a similar number of ROIs and calcium traces. About 400 ROIs, uniformly covering the field of view, were typically identified per recording. The average fluorescence Fi (t) in each ROI i along the recording was then extracted, corrected from global drifts and artifacts, and finally normalized as (Fi (t) − F(0,i)) / F(0,i) = fi (t), where F0,i is the background fluorescence of the ROI. The time series of fi (t) was analyzed with NETCAL to infer neuronal activation timing using the Schmitt trigger method. Our analysis used + 2 S.E.M. of the baseline noise as the high threshold, + 1.5 S.E.M. as the low threshold, and 200 ms as the minimum event length. Calcium traces were calculated, and raster plots of network activity were then constructed by representing the trains of detected neuronal spikes over time. Next, network bursts were analyzed to quantify the ability of the neuronal networks to exhibit collective dynamics, i.e., the collective activation of a group of neurons in a short time window. Bursts were investigated using two approaches. In the first approach, raster plots of spike events were scanned to detect collective occurrences in which at least 5% of the neurons in the network fired synchronously within a 500-ms window. This threshold of 5% was set to disregard random activations that coincided in time. In the second approach, the fluorescence time series of all neurons in the network were averaged. The resulting trace was analyzed with the Schmitt trigger method to detect sufficiently strong fluorescence peaks associated with bursting episodes. Both approaches produced consistent results. Although the detected bursts contained a different number of participating neurons, this information was disregarded in the present analysis and treated later. The total number of detected network bursts divided by the recording duration reflected the culture’s activity and was indicated as bursts/min.

The fraction of active neurons in the network was calculated as follows. All detected ROIs were assigned as neurons. After inferring the spike trains, those neurons exhibiting at least two spikes along the recording were considered active, and their number NA was set as a proxy of the healthy population in the neuronal network. The average fraction of active neurons in each condition was then determined as f = NA/NT, where NT is the total number of detected ROIs.

At least ten videos of each genotype/day from different culture plates were consecutively analyzed. Data are presented as the mean of network burst/min ± S.E.M. Statistical analysis was performed using two-way ANOVA + Bonferroni’s multiple comparisons test (GraphPad Prism 8 software). Asterisks indicate significant differences between Prnp+/+ and PrnpZH3/ZH3 cultures at a given DIV: *p < 0.05 and ***p < 0.001. The # symbols indicate significant differences between a given DIV with the initial value at 8 DIV: ###p < 0.001.

Statistical analysis

All statistical analysis was performed with GraphPad PRISM 8 (GraphPad Software, USA). Unless otherwise stated, data is plotted as the mean ± SEM. All experiments were performed three times unless specified. Normality of the distributions was checked via Shapiro-Wilk test. All tests performed were two-sided, and adjustments for multiple comparisons and/or significantly different variances (Fisher’s F) applied were indicated. All data analysis was performed blind to the experimental group by two independent experimenters. Unless otherwise stated, sample size was chosen in order to ensure a power of at least 0.8, with a type I error threshold of 0.05, in view of the minimum effect size that was expected.

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