Interneuronal GluK1 kainate receptors control maturation of GABAergic transmission and network synchrony in the hippocampus

Animals

Experiments were performed using the following mouse lines: Grik1tm1c/tm1cand Gad2-Grik1tm1d/tm1d. Grik1tm1a (KOMP)Mbp mice in C57BL/6N background were obtained from KOMP repository (UC Davis) and crossed with CAG-Flp transgenic line to produce a floxed conditional knock-out mice (Grik1tm1c/tm1c) [20]. The Grik1tmc1/tm1c mice were crossed with Gad2tm2(cre)Zjh/J mice (expressing Cre under the Gad2 promoter) to obtain the Gad2-Grik1tm1d/tm1d line (referred to as Gad-Grik1−/−). Homozygous animals were used for all the in vitro experiments. Grik1tm1c/tm1c mice heterozygous for the Gad2-Cre, with littermate Grik1tm1c/tm1c controls were used for the behavioral and in vivo experiments. All the experiments were done in accordance with the University of Helsinki Animal Welfare Guidelines and approved by the Animal Experiment Board in Finland.

In vitro electrophysiology

Preparation of acute slices. Acute sagittal sections (300–400 μm) were prepared from brains of neonatal (P4–P6) male or female mice, juvenile (P18–P21) or adult (> P50) male mice using standard methods. Briefly, mice were decapitated under isoflurane anesthesia, the brains were extracted and immediately placed in carbogenated (95% O2/5% CO2) ice-cold sucrose-based dissection solution containing (in mM): 87 NaCl, 3 KCl, 7 MgCl2, 1.25 NaH2PO4, 0.5 CaCl2, 50 sucrose, 25 glucose, 25 NaHCO3 (in majority of the experiments) or in low Ca2+–high Mg2+ dissection solution, containing (in mM): 124 NaCl, 3 KCl, 1.25 NaH2PO4, 26 NaHCO3, 15 glucose, 10 MgSO4, 1 CaCl2 (part of experiments in the neonatal mice). The hemispheres were separated and slices were cut using a vibratome (Leica VT 1200S). Slices containing the hippocampus were placed into a slice holder and incubated for 30 min in carbogenated, warm (34 °C) High-Mg2+ ACSF (in mM): 124 NaCl, 3 KCl, 1.25 NaH2PO4, 26 NaHCO3, 15 glucose, 3 MgSO4*7H2O, 2 CaCl2. Slices were then maintained at room temperature.

Electrophysiological recordings from acute slices. After 1–5 h of recovery, the slices were placed in a submerged heated (32–34 °C) recording chamber and perfused with standard ACSF, containing (in mM): 124 NaCl, 3 KCl, 1.25 NaH2PO4, 26 NaHCO3, 15 glucose, 1 MgSO4, 2 CaCl2 (95% O2 / 5% CO2) at the speed of 1–2 ml/min. Whole-cell patch-clamp recordings were done from CA3 principal neurons under visual guidance using patch electrodes with resistance of 2–7  MΩ, filled by low-chloride filling solution, containing (in mM): 135 K-gluconate, 10 HEPES, 2 KCl, 2 Ca(OH)2, 5 EGTA, 4 Mg-ATP, and 0.5 Na-GTP, 280–285 mOsm (pH 7.2–7.35. Multiclamp 700B amplifier (Molecular Devices), Digidata 1322 (Molecular Devices) or NI USB-6341 A/D board (National Instruments) and WinLTP version 2.20 or pClamp 11.0 software were used for data collection, with low pass filter (6 kHz) and sampling rate of 5 or 20 kHz. In all voltage-clamp recordings, uncompensated series resistance (Rs) was monitored by measuring the peak amplitude of the fast whole-cell capacitance current in response to a 5 mV step. Only experiments where Rs < 30 MΩ, and with < 20% change in Rs during the experiment, were included in analysis. The drugs were purchased from Tocris Bioscience (ACET: 2728, ATPA: 1107).

Preparation of organotypic hippocampal cultures on MEA probes. Before use, MEA probes (MED-P515A, Alpha MED Scientific, 8 × 8 electrodes; electrode size 50 × 50 μm; array size 1 × 1 mm; spacing 150 μm) were sterilized with 70% ethanol for one hour, washed with sterile water and dried under UV-light for one hour. For adhesive coating, the probes were treated with sterilized poly-L-lysine (Sigma-Aldrich) diluted 1:10 with MQ-water (1 ml dilution/probe) over night at RT. Next day, before starting culturing, the probes were washed 3 times with sterile MQ-water.

P4-P5 mice were quickly decapitated, the whole head was briefly immersed in 70% EtOH and transferred into a laminar hood in Gey′s Balanced Salt Solution (GBSS) (G9779, Sigma). The brain was extracted, the hemispheres were separated and placed on a stage, and covered with 1–2 ml of low melting point agarose gel. 350 µm coronal slices were cut using a tissue chopper (McIlwain), the slices were placed in cold GBSS and the hippocampus was extracted from the slices. The hippocampi were placed on Poly-L-lysine coated MEA probes with 280 µl preheated Neurobasal A (Gibco) medium, supplemented with 2% B27-supplement (Gibco), 2 µM L-glutamine and chloramphenicol (NB-A medium). The medium was changed and the probes were put in a petri dish containing 1–2 ml sterile H20, and placed in the humidified cell culture incubator (+ 37 °C, CO2 5%), first 60 min without rocking and then on a slowly moving plate rocker. The medium was added or changed in the cultures according to the following plan: Days in Vitro (DIV) 0: 280 µl NB-A, DIV 1: add 20–60 µl NB-A, DIV 2: change ~ 70% NB-A, 3 DIV 2: add 20–60 µl NB-A, DIV 4: change ~ 50% BrainPhys (BrainPhysTM Neuronal medium supplemented with SM1 neuronal supplement (Stemcell Technologies), and 0.5 mM L-glutamine), followed by adding 20–60 µl BrainPhys or changing ~ 70% BrainPhys on alternating days until 10 DIV.

Electrophysiological recordings from cultured organotypic hippocampus slices. The spontaneous activity of the slices was recorded at four time points, at DIV5, 7, 9 and 10. Before recording, on 5, 7, and 9 DIV 20–60 µl fresh medium was added and 10 DIV 70% medium was changed, and the cultures were left for minimum one hour in the incubator before starting the recording. The slices on the probes were then imaged using Leica MZ10F microscope (Leica Microsystems GmbH, Wetzlar, Germany) and Qimaging Rolera Bolt Scientific camera (Teledyne QImaging, BC, Canada) to ensure healthy morphology and to confirm electrode positions. Slices that had detached from the probe or had clear, visually detectable damage such as holes in the tissue were rejected. The probes were connected to the MED64 system by the MED-C03 connector inside the recording incubator (+ 37 °C, CO2 5%). The signal was monitored for 30 min for stabilization, followed by 10–30 min of recording using the MED64-amplifier (MEDA64HE1) and Mobius software (Alpha MED Scientific), with 20 kHz sampling rate, 0.1 Hz high pass and 5 kHz low pass filtering and 5 mV voltage range.

In vivo electrophysiology

Head plate implantation surgery. The mice were anesthetized using ≥ 4% isoflurane. Carprofen (Rimadyl vet, 5 mg/kg s.c.), dexamethasone (2 mg/kg s.c.) and buprenorphine (Bupaq vet, 0.05 mg/kg s.c.) were injected to reduce pain, inflammatory response and brain edema. The mice were then placed on a 37 °C heating pad in the stereotaxic device. The depth of anesthesia was adjusted to 1.5–2% isoflurane and both eyes were covered with ophthalmic ointment (Viscotears, Novartis) to protect the cornea from dehydration. The hair on top of the head was trimmed and the skin was disinfected by povidone-iodine (Betadine). Lidocaine (0.5%, max 5 mg/kg) was injected under the scalp for local anesthesia, and scalp and periosteum were removed. The surface of the skull was roughened using a large diameter drill bit and carefully cleaned. Tissue adhesive (3 M Vetbond) was used to seal the edges of the wound. The locations for craniotomies were identified and marked using a drill. The lightweight stainless-steel head plate with round 8.2 mm opening (Neurotar model 5) was attached to the skull using a small amount of super glue (Loctite precision), and the sides of the head plate were covered with dental cement (3 M RelyX). After operation the animals were allowed to recover on the heating pad, and placed in the home cage with some water-soaked soft food pellets. The weight of the mice was monitored and carprofen (Rimadyl vet, 5 mg/kg s.c.), and buprenorphine (Bupaq vet, 0.05 mg/kg s.c.) were injected for two days after the surgery for post-operative care. In case of weight loss, 0.1–0.3 ml glucose (20 mM; max: 20 ml/kg) solution in saline was i.p. injected in the first 3 days after operation.

Habituation, craniotomy and electrophysiological recording from awake, head-fixed mice. After recovery from the head-plate surgery (at least 3 days), the mice were habituated to being head-fixed for 5 min, 10 min, 15 min, 30 min and 1 h on consecutive days before the recordings. On the day of recording or one day before, a small craniotomy was made for placing the recording probe. Briefly, the mice were anesthetized using ≥ 4% isoflurane. Carprofen (Rimadyl vet, 5 mg/kg s.c.) was injected, and the mice were placed on a 37 °C heating pad in the stereotaxic device. The depth of anesthesia was adjusted to 1.5–2% isoflurane and both eyes were covered with ophthalmic ointment (Viscotears, Novartis). A grounding screw for connecting the ground electrode was attached to the skull using dental cement (3 M RelyX). A small circular craniotomy was drilled in the right hemisphere on top of the recording site (RC: (− 2)–(− 2.1) mm, ML: 0.9–1.3 mm for CA1 and DG; RC: (− 2.1)–(− 2.3) mm, ML: 2.3–2.6 mm for CA3) and covered with silicone adhesive (Kwik-Sil, World Precision Instruments). A bath from Kwik-Sil with ca 10 mm high walls was constructed on top of the head plate opening. The mice were let to recover in the home cage for at least one hour prior the recording.

For recording, the mice were head-fixed on top of a rotating ball. The ground electrode was attached to the grounding screw and a linear probe (Neuropixels 1.0) was placed on the surface of the brain and inserted horizontally into the desired depth (3.5 mm for CA1 and DG, 3.7–4.0 mm for CA3). Before insertion the probe was stained by the DiI (V22885, TermoFisher) droplet, to allow post-experimental histological evaluation. The bath on top of the head plate was filled with sterile filtered saline (0.9% NaCl). After 15 min stabilization period, simultaneous recordings of local field potential (LFP) and multi-unit activity (MUA) were performed at 30 kHz sampling rate (2500 Hz online down sampling for the LFP signal) using the IMEC Neuropixels acquisition system and Spike GLX software. A custom made Python application was used for video acquisition. After the recording, the craniotomy was covered with Kwik-Sil. The recordings for CA1/DG and CA3 were done on consecutive days. Within one week from recording the mice were transcardially perfused by PBS followed by 4% PFA and the brains were collected for histological analysis.

Data analysisAnalysis of the in vitro electrophysiological recordings

The frequency and amplitude spontaneous IPSCs (sIPSCs), EPSCs (sEPSCs) and network bursts were analyzed using Minianalysis 6.0.0.3 (Synaptosoft). For the sIPSCs (outward current) and sEPSCs (inward current), the amplitude threshold was set two times the baseline RMS noise level. Detected events were verified manually. Network bursts were identified on the base of slow outward current, with an amplitude and duration of at least 10 pA and 100 ms, respectively. For sIPCSs and sEPSCs, at least 5 min per condition or 200 events were analyzed, and the network bursts were analyzed from the complete recording. Cells with spontaneous event frequency less than 0.008 Hz (0.5 event/min) were excluded.

For the MEA data analysis, representative channels were selected from the CA1, CA3 and DG regions based on the images taken before data acquisition. Spikes and bursts (minimum number of spikes = 10, minimum duration of burst = 0.1 s) from the MEA recordings were detected using NeuroExplorer 5.205 functions DetectSpikes (using default parameters) and Bursts (using the Surprise algorithm with default parameters). Dentate spikes for MEA activity spread analysis were detected using Spike2 (v. 9.03a Cambridge Electronic Design), and cluster activity following the DG spikes was detected using a custom made Matlab script. Before a calculation of activity clusters traces were downsampled (5 kHz) and demeaned (1 s window with 50% overlap). We defined an activity cluster as a spatially adjacent (bwconncomp MATLAB function) group of channels in which local field potential is higher (for positive clusters) or lower (for negative clusters) than a threshold value (2 and − 2 standard deviation of the signal, respectively). Activity clusters were calculated in 21 time points near each spike (100 ms window centered at the spike timestamp with 5 ms time lag). If there were several clusters at one time point, only a cluster with a maximum size was used for further calculations.

Detection of epochs of rest and movement

Deep Lab Cut 2.2.0.2 [76, 77] was used to track 7 points (the front paws, the mouth, and the tip, philtrum and nares of the snout) in the videos recorded during the in vivo data acquisition. We used ResNet-50-based neural network [78, 79] to train the model for detection and tracking of the mouse body parts. The DLC tracking data was passed to SimBA 1.3.12 [80] to detect epochs of movement and rest. The SpikeGLX command line tool CatGT (v. 2.4) was used to extract event times from the video TTL signal that was recorded to synchronize video frames to the electrophysiological signals. A custom-made Python program was used to extract the electrophysiological data corresponding to each detected epoch.

Analysis of oscillatory power and cross-frequency coupling

The data was processed using custom-made Python programs, with MNE (v. 0.24.0), Numpy (v. 1.19.5), ripple_detection (v.1.2.0) modules and readSGLX from the SpikeGLX_Datafile_Tools package. The signals were low-pass filtered to 350 Hz and DC-components were removed by subtracting channel means. For each epoch, a time–voltage–plot covering all channels was plotted, and epochs with artifacts or noise were manually discarded based on the plotted figures.

Channels for analysis were selected on the basis of post-mortem histological investigation (i.e. recording sites of fluorescently-marked electrodes), LFP signal properties (i.e. amplitude, phase shift, CA1 ripples) and visually identified spike patterns. 10–20 channels representing (i) the CA1 stratum pyramidale, CA1 stratum moleculare and Dentate Gyrus (DG) regions and (ii) the CA3 stratum pyramidale and CA3 stratum moleculare were selected for analysis. The recorded signals in selected channels were divided into 3 s epochs of idle and running based on video analysis of the mouse activity.

For each sub region separately, complex Morlet wavelet convolution was performed using the MNE tfr_array_morlet-function, separately for theta (3–12 Hz) and gamma (20–90 Hz) frequencies, and the mean power on each frequency band was extracted for all epochs. For the theta phase—gamma power intermodulation analysis, the theta phase at 7 Hz was extracted and divided into eight equal sized phase angle bins. The average gamma (20–90 Hz) power corresponding to each theta phase bin was then calculated. Ripple oscillation in the CA1 pyramidal layer were detected using the Kay_ripple_detector method [36] from the ripple_detection package from Eden-Kramer Lab [81].

Behavioral tests

Open field The behavioral apparatus consisted of four 50 cm × 50 cm arenas (light grey PVC, with wall height of 40 cm) placed under camera for tracking the movement of animals by Ethovision XT15 (Noldus). The illumination was applied by indirect diffuse room light (20–25 lx). Each animal was released in one of the corners and monitored for 10 min. The mice were then removed to the holding cage and a 12 cm × 4 cm semi-transparent 50 ml falcon tube was placed in the center of each arena. The animals were then released in the arena and observed for additional 10 min.

Elevated plus-maze (EPM) The maze consisted of two open arms (30 × 5 cm) and two enclosed arms (30 × 5 cm, inner diameter) connected by central platform (5 × 5 cm) and elevated to 40 cm above the floor. The floor of each arm was light grey and the closed arms had transparent (15 cm high) side- and end-walls. The illumination level in all arms was ~ 150 lx. The mouse was placed in the center of the maze facing one of the enclosed arms and observed for 5 min. The latency to the first open arm entry, number of open and closed arm entries (four paw criterion) and the time spent in different zones of the maze were measured. The number of faecal boli was counted after trial. Distance travelled and time spent in different areas (open, closed) was recorded with Ethovision XT 10 tracking equipment (Noldus, Netherlands).

IntelliCage (IC) Mice were subcutaneously injected with RFID transponders (Planet ID GmbH, Germany) for individual identification. The IntelliCage by NewBehavior (TSE Systems, Germany) is an apparatus designed to fit inside a large cage (610 × 435 × 215 mm, Tecniplast 2000P). The apparatus itself provides four recording chambers that fit into the corners of the housing cage. Access into the chambers is provided via a tubular antenna (50 mm outer and 30 mm inner diameter) reading the transponder codes. The chamber contains two openings of 13 mm diameter (one on the left, one on the right) which give access to drinking bottles. These openings are crossed by photo beams recording nose-pokes of the mice and the holes can be closed by motorized doors. Four triangular red shelters (Tecniplast, Buguggiate, Italy) were placed in the middle of the IntelliCage and used as sleeping quarters and as a stand to reach the food. The floor was covered with a thick (2–3 cm) layer of bedding. The IntelliCage was controlled by a computer with dedicated software, executing preprogrammed experimental schedules and registering the number and duration of visits to the corner chambers, nose-pokes to the door openings and lickings as behavioral measures for each mouse. In the beginning of the test, the mice were released in the IntelliCage with all doors opened allowing unlimited access to the bottles (free adaptation).

Adaptation to nose-poke All doors were closed at the beginning of experiment and mice were required to poke into closed gates to reach drinking tubes. Only the first nosepoke of the visit opened the door for 5 s (pre-defined time). Animals had to start a new visit in order to get access to water again.

Adaptation to drinking sessions Doors were programmed to open after the first nose-poke only during two 2-h periods, from 20:00 to 22:00 and from 04:00 to 06:00. Drinking sessions were applied for increasing the motivation to visit the corners and thereby providing defined time windows for testing of learning.

Flexible sequencing task The animals were assigned two correct corners, which were rewarded alternately during drinking sessions (task acquisition—after visiting a correct corner, the next reward could be obtained in diagonally opposite corner, correct sequence of visits 1–3-1–3 etc., corners 2 and 4 were assigned as incorrect and never rewarded). After 4 days (8 sessions) the sequence was reversed, i.e. reward (water) was delivered in previously incorrect corners for next 8 sessions. After first reversal session, two more reversals were performed.

Home cage activity Male Control and Gad-Grik1−/− were housed individually in cages with an infrared sensor (InfraMot; TSE-Systems) to monitor their activity over 6 days (excluding the first night of adaptation) with 12/12 h dark/light cycle. The mean hourly activity during the dark/active phase of mice was averaged over the 6 days.

Barnes maze (BM) The maze consists of a circular platform (100 cm diameter) with 20 holes (5 cm diameter) around the perimeter (Ugo Basile, Italy). One of the holes was connected with a dark chamber filled with bedding material and two food pellets, the escape box. Two days before the experiment, each animal was introduced to the escape box for 2–3 min. The bright light (500–600 lx on the platform) was used to motivate the mice to find and enter the escape box. The mice were trained to find the escape box in three (day 1–2) or two (day 3) training trails per day (inter-trial interval at least 60 min) over three days. The training trial ended when the mouse entered the escape box or after 3 min as cut-off time (in this case, the mouse was gently directed to the escape box). The memory test was carried out during the first trial on day 4 when the mice were monitored on the platform without escape box for 90 s. Thereafter, reversal learning was carried out, where the escape box was moved under the opposite hole and the mice received two training trials on day 4 and 5. After the last training trial on day 5, the second memory test was performed. Throughout the testing, the movement of animals was tracked by Ethovision XT15 (Noldus).

Statistical analysis

All data was transferred to GraphPad Prism (v. 9.3.1.471 or v. 9.4.1.681) for statistical analysis. The basal frequency of sIPSCs and of sEPSCs in control and Gad-Grik1−/− pyramidal CA3 cells across different age groups (neonatal, juvenile and adult) were analyzed by 2-way ANOVA. To compare the event frequencies between the genotypes within each age group we used multiple Mann–Whitney test as a post-hoc, as the model residuals were not normally distributed. The basal frequencies of the spontaneous network bursts in control vs Gad-Grik1−/− neonatal hippocampus were normally distributed and compared by unpaired two-tailed t-test test. For testing of drug effects (application vs baseline), two-tailed paired t-test or Wilcoxon match-pairs sign-rank test were used, for normally or not normally distributed model residuals, respectively. All the statistical tests were performed on raw data. For the graphical representation of the drug effects, the frequency during the drug application was normalized to that during the baseline recording.

The spike frequency, burst duration and frequency, and activity spread clusters in the MEA data were analyzed by 2-way ANOVA. To compare the activity spread during 0–20 ms and 30–70 ms after DG spike we used the Mann–Whitney U-test. For the in vivo data, we used 2-way ANOVA to compare the theta and gamma power, ripple frequencies and durations, and sum-of-squares F-test to test the quadratic curve fit. We used 2-way ANOVA to compare the groups and Holm-Šídák’s test as post-hoc for multiple pairwise comparisons in the behavioral experiments. We used two-tailed unpaired t-test for comparing the relearning slopes calculated for the Barnes Maze experiments.

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