For ex vivo electrophysiology recordings, 4–8 weeks old male RjOrl:SWISS mice were used, with the exception of one experiment that was performed in 4–8 weeks old male C57Bl/6 mice, as noted in the results section. Experiments were conducted according to the policies on the care and use of laboratory animals stipulated by the ministries of research of the different countries in compliance with the European Communities Council Directive (2010/63). All efforts were made to minimize animal suffering and reduce the number of animals used. The animals were housed three to six per cage under controlled laboratory conditions with a 12-h dark-light cycle and temperature of 22 ± 2 °C. Animals had free access to standard rodent diet and tap water.
For in vivo LTP experiments, all injections and recordings were performed on adult male Sprague-Dawley rats (450–650 g). The experiments were performed in accordance with local institutional and governmental regulations regarding the use of laboratory animals at the University of Frankfurt as approved by the Regierungspräsidium Darmstadt and the animal welfare officer responsible for the institution.
For in vivo calcium imaging experiments, male and female C57Bl/6 mice (~P40) were used. Experiments were conducted in compliance with institutional (Technische Universität München) animal welfare guidelines and approved by the state government of Bavaria, Germany.
Synthetic Aη peptides were obtained from Peptide Specialty Laboratories (PSL GmbH; Heidelberg, Germany) and consisted of the following sequences:
Synthetic Aη–α (sAη–α, 108 amino acids) sequence:
Synthetic Aƞ–β (sAη–β, 92 amino acids) sequence:
Synthetic N-term sAη (sAη–NT, 46 amino acids) sequence:
Synthetic C-term sAη–β (sAη–β–CT; 46 amino acids) sequence:
The peptides were dissolved in dimethyl sulfoxide (DMSO) at 100 μM and placed at − 80 °C for long-term storage. For ex vivo electrophysiology, on day of experiment, aliquots were further diluted in artificial cerebrospinal fluid (aCSF) (see below) to the required concentration (1–100 nM).
For in vivo electrophysiology, aliquots were further diluted on day of experiment in phosphate buffered saline (PBS) to the required concentration (1 μM).
For in vivo calcium imaging, the peptides were combined with Ringer’s solution (see below) on day of experiment to 100 nM.
Recombinant Aη peptides were generated and purified as described previously . Briefly, for the expression of Aη-α and Aη-β in CHO cells, the complementary cDNAs of the respective fragments were amplified by PCR and subcloned into the pSecTag2A vector (Invitrogen) that features an N-terminal secretion signal. CHO cells were cultured in DMEM with 10% FCS and non-essential amino acids. Transfections were carried out using Lipofectamine2000 (Invitrogen) according to the manufacturer’s instructions. The next day media was changed to OPTIMEM (Invitrogen) and the serum-free conditioned media of the transfected cells, expressing the recombinant Aη peptides, were collected after 20 h. Up to 1 l of C-terminally HIS-tagged peptides was collected and filtered (0.2 μM; Tabletop filter from Millipore). The filtrate was purified by anion exchange chromatography using HiTrap columns for small-scale protein purification on Äkta system (Cytiva; Ni-NTA). Positive fractions were pooled and the elution buffer was exchanged and concentrated using an Amicon Ultra Centrifugal filter (PLBC Ultracel-PL membrane, 3 kDa MWCO) with 3 volumes of aCSF. The protein concentration was measured based on the OD280 with a Nanodrop device (Thermo Fisher) and calculated for each protein based on the molecular weight of the nonglycosylated peptide including the myc-HIS tag. The preparation was diluted to a final concentration of 10 nM in ACSF on the day of the experiment.
Biochemical analysis of Aη peptides
Protein concentration of the purified peptides was measured with a Nanodrop spectrophotometer (Thermo Fisher Scientific, Germany) and the molar concentration was calculated and adjusted according to the molecular weight of the peptides (recAη-β MW: 13120.43; recAη-α MW: 15057.45). Peptides were stored until use in a − 80 °C freezer. For quality control, 1 μg of recombinant proteins and synthetic peptides were separated on a Tris-Tricine gel (10–20%, Thermo Fisher Scientific, Germany), stained with GelCode Blue stain, and imaged with an ImageQuant 800 system (Amersham, Germany).
Ex vivo electrophysiology
Mice were culled by cervical dislocation and hippocampi were dissected and incubated for 5 min in ice-cold oxygenated (95% O2/5% CO2) cutting solution (in mM): 206 sucrose, 2.8 KCl, 1.25 NaH2PO4, 2 MgSO4, 1 MgCl2, 1 CaCl2, 26 NaHCO3, 10 glucose, 0.4 sodium ascorbate, oxygenated with 95% O2 and 5% CO2 (pH 7.4). Hippocampal slices (350 μm) were cut on a vibratome (Microm HM600V, Thermo Scientific, France). For recovery, slices were then incubated in oxygenated aCSF for 1 h at 37 ± 1 °C and then stored at room temperature until used for recordings. aCSF composition was (in mM) 124 NaCl, 2.8 KCl, 1.25 NaH2PO4, 2 MgSO4, 3.6 CaCl2, 26 NaHCO3, 0.4 sodium ascorbate, 10 glucose, oxygenated with 95% O2 and 5% CO2, and pH 7.4. All chemicals were from Sigma-Aldrich (Saint-Quentin Fallavier, France). Recordings for all experiments were done at 27 ± 1 °C in a recording chamber on an upright microscope with IR-DIC illumination (SliceScope, Scientifica Ltd., UK). Field recordings were performed using a Multiclamp 700B amplifier (Molecular Devices, San Jose, CA, USA), under the control of pClamp10 software (Molecular Devices, San Jose, CA, USA). Data analysis was executed using Clampfit 10 software (Molecular Devices, San Jose, CA, USA). Field excitatory post-synaptic potentials (fEPSPs) were recorded in the stratum radiatum of the CA1 region (using a glass electrode filled with 1 M NaCl and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4). The stimuli were delivered to the Schaffer collateral pathway by placing a monopolar glass electrode (capillary Glass, 1.5 mm outer diameter, 0.84 mm inner diameter, WPI, France, filled with aCSF) in the stratum radiatum. fEPSP response was set to approximately 30% of the maximal fEPSP response i.e. approx. 0.2–0.3 mV, with stimulation intensity 10 μA ± 5 μA delivered via stimulation box (ISO-Flex, A.M.P.I. Inc., Israel). Electrodes were placed superficially to maximize exposure to peptides. Slices were perfused with oxygenated aCSF. The baseline fEPSP was obtained by stimulating at 0.066 Hz (1 stimulation/ 15 s). A stable baseline of a fEPSP was first obtained in control conditions (at least 10 min). Then, synthetic or recombinant peptides were applied for at least 15 minutes (for 100 nM data) or 20 min (for other peptide concentrations) to ensure consolidation of baseline prior to LTP induction. If the baseline was not consolidated within 45 min after peptide application, the slice was discarded. Upon confirmation of this stable baseline, LTP was then induced. The peptide was also recirculated throughout the 1-h recording after induction. LTP was induced by a high-frequency stimulation (HFS) protocol: 2 pulses at 100 Hz for 1 s with a 20-s inter-stimulus interval (ISI). “Control” LTP experiments (aCSF only, no application of peptide) were routinely performed interleaved with peptide application during the same experimental period (i.e., same experimental batch on the same batch of mice, on the same electrophysiology rigs, by the same experimenter within the same continuous timeframe).
For all LTP recordings, only the first third of the fEPSP slope was analyzed to avoid population spike contamination. For LTP time-course and bar graph analyses, the first third of the fEPSP slope was calculated in the baseline condition and at 45–60 min post-induction in each recording. The average baseline value was normalized to 100% and values at 45–60 min post LTP induction were normalized to this baseline average (1-min bins).
For paired-pulse ratios (PPRs), two stimuli were delivered at 100, 200, and 300 ms inter-stimulus intervals (ISI). PPRs were calculated as the average of fEPSP2 slope/fEPSP1 slope (10 sweeps average per ISI). Recordings of control (aCSF only) and peptide conditions were interleaved within the same day.
The input/output (I/O) curves were generated by calculating the fEPSP slope corresponding to a given fiber volley (FV) amplitude ranging from 0.1 to 0.4 mA in increments of 0.1 mA measuring 10 sweeps averages. This protocol was first done under aCSF and slices then perfused for 20 min in aCSF either with or without the peptide before repeating the protocol, as within slice control. Input/output graphs compared the fEPSP slope corresponding to the fiber volley measurements at both time points.
In vivo electrophysiology
Urethane (Sigma-Aldrich GmbH, Munich, Germany) solution was used to anesthetize the animals with an initial injection (2 g/kg body weight) applied intraperitoneally. Supplemental doses (0.2–0.5 g/kg) were injected subcutaneously until the interdigital reflex could no longer be triggered. The body temperature of the animal was constantly controlled through a rectal probe and maintained at 36.5–37.5 °C using a heating pad. For local anesthesia of the scalp, prilocaine hydrochloride with adrenalin 1:200,000 (Xylonest 1%, AstraZeneca GmbH, Wedel, Germany) was injected subcutaneously at the site of incision. The head of the anesthetized rat was placed into a stereotaxic frame for accurate insertion of electrodes and injection cannula. Using standard surgical procedures, we drilled the stimulation and recording holes and removed the dura mater. A tungsten recording micro-electrode glued to a 10-μl Hamilton series syringe was lowered unilaterally into the dentate gyrus hilus (2.5 mm lateral and 3.8 mm posterior to bregma), and a bipolar concentric stimulating electrode (World Precision Instruments, Germany) was lowered unilaterally into the perforant path (4.5 mm lateral to lambda), while monitoring the laminar profile of the response. Current pulses (30–800 μA, 0.1–0.2 ms duration) were generated by a stimulus generator (STG1004, Multichannel Systems, Reutlingen, Germany). The recorded fEPSPs were first amplified (P55 preamplifier, Grass Technologies, West Warwick, RI, USA) and then digitized at 10 kHz for visualization and offline analysis (Digidata 1440A, Molecular Devices, San Jose, CA, USA). The analysis of electrophysiological data was executed using Clampfit 10.2 software (Molecular Devices, San Jose, CA, USA) as well as custom MATLAB scripts (The MathWorks, Natick, MA, USA). As a measure of synaptic LTP, we compared responses with baseline stimulation (at 0.1 Hz) prior to theta-burst stimulation (TBS) with responses subsequent to TBS. At the start of each experiment, stable baselines were recorded. Then, the experimental solution was injected into the hippocampus. In each experiment, injections of 1 μl of sAη–α or sAη–β–CT (1 μM) were delivered from the Hamilton syringe attached to a microinjection unit (Model 5000, Kopf Instruments, Tujunga, CA, USA). Intradentate injections of fluid led to a typical temporary reduction in the fEPSP slope and amplitude, probably caused by changes in extracellular resistivity . The degree of response suppression and recovery can be seen in LTP graphs. After a baseline period of 20 min, LTP was induced using a standard TBS protocol: six series of six trains of six pulses at 400 Hz, with 0.2 s between trains and 20 s between series. Both the pulse width and the stimulus intensity during TBS were doubled in comparison to baseline recordings. The LTP was followed for 60 min using the baseline stimulation protocol. For the analysis of the slope of the fEPSP, only the early component of the waveform, which is not affected by the population spike, was used. LTP in Fig. 5b, c was calculated as an average % baseline of fEPSP slope for the first 10 min (1–10 min) or last 11 min (50–60 min) post TBS. The potentiation of the fEPSP slope was expressed as a percentage change relative to the pre-TBS baseline.
In vivo multiphoton calcium imaging
The procedure for animal preparation followed the same protocol as described previously [3, 9]. Briefly, C57Bl/6 mice were placed in an induction box and anesthetized using isoflurane (~ 3–4%). Following induction, animals were transferred to a stereotaxic frame and heating plate (37–38 °C) and maintained using 1–1.5% isoflurane during surgical procedures, with respiration and pulse rate continuously monitored. The skin was first carefully excised and retracted, and a custom-made recording chamber/well affixed to the exposed skull. Subsequently, a small craniotomy (1 mm2, 2.5 mm posterior to bregma, 2.2 mm lateral to the midline) was performed and the exposed cortical tissue carefully aspirated to reveal the underlying hippocampus (CA1). The recording chamber was perfused with warmed Ringer’s solution (in nM): 125 NaCl, 4.5 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2 and 20 glucose, pH 7.4, 95% O2 and 5% CO2, and the hippocampus stained using Fluo-8®, AM (0.6 mM) (AAT Bioquest, Inc., Sunnyvale, CA, USA) via the multi-cell bolus loading injection technique . Peptides (100 nM) were perfused into the recording chamber for bath application to the exposed CA1 region of the hippocampus (45–60 min wash in). In vivo imaging was conducted as described previously using a custom-built two-photon microscope consisting of a titanium:sapphire laser (coherent; λ=925 nm), resonant scanner, a Pockels cell laser modulator, and a water-immersion objective (Nikon; 40 × 0.8 numerical aperture) . Images were acquired at a sampling rate of 30 Hz using custom-written LabVIEW routines and analyzed off-line in LabVIEW (National Instruments, Austin, TX, USA), Igor Pro (WaveMetrics, Inc., Lake Oswego, OR, USA), and MATLAB (The MathWorks, Natick, MA, USA). Regions of interest (ROIs) were manually defined around individual cell bodies, and time series of relative calcium fluorescence changes (ΔF/F) were extracted for each ROI. Significant changes in fluorescence were defined as ΔF/F calcium transients which exceeded background noise levels by > 3 standard deviations (SD), in accord with the analytical approach used in our previous publication studying Aη peptides using multiphoton calcium imaging  and for direct comparison with the current study. Animals were maintained at low levels of isoflurane anesthesia (~ 0.8%) throughout imaging procedures.
Detailed statistics are presented in supplementary Tables S1, S2, S3, S4 and S5. Results are shown as mean ± S.E.M. Significant effects were inferred at p< 0.05.
For ex vivo and in vivo electrophysiology, statistical analyses were performed with Prism GraphPad 6.0 Software (GraphPad Software, La Jolla, CA, USA). “N” refers to the number of animals and “n” to the number of slices examined. For ex vivo electrophysiology data analysis, each peptide condition was plotted and analyzed against its own interleaved controls performed within the same experimental period. The normality of data distribution was verified with Shapiro-Wilk’s test. When normally distributed, an unpaired Student’s two-tailed t-test was used for comparison of two independent samples. When normality was not observed, a Mann-Whitney test was used for comparison of two independent samples. For comparison of more than 2 conditions, a one-way or two-way ANOVA was used followed by Dunnett’s test or Sidak’s tests for post hoc comparisons, as appropriate.
Statistical analyses of in vivo calcium imaging data were performed using MATLAB (The MathWorks, Natick, MA, USA). Following testing for normality using a one-sample Kolmogorov-Smirnov, the non-parametric Mann-Whitney U test was used to test for equality of population medians. For statistical testing of more than two groups, the nonparametric Kruskal-Wallis test was used with Tukey-Kramer correction for multiple comparisons. The experimenter was blinded to the synthetic peptide being administered and corresponding data only decoded at the end of experimentation.