Synaptic organisation and behaviour-dependent activity of mGluR8a-innervated GABAergic trilaminar cells projecting from the hippocampus to the subiculum

Phaseolus vulgaris leucoagglutinin (PHAL; Vector Laboratories; 2.5% in 0.1 M PB solution) was iontophoretically injected (Gerfen and Sawchenko 1984) using a glass pipette with tip diameter of 12–18 μm into the medial septum of rats and mice (stereotaxic coordinates relative to Bregma: in rat, 0.6 mm anterior, 1.4 mm lateral and 5 mm, 5.5 mm and 6 mm ventral with 15° angle; in mouse, 0.85 mm anterior, 0 mm lateral and 3.6 mm ventral with 0° angle). Positive current pulses of 5 μA were applied every 7 s for 15–30 min. To minimise tissue damage and dorsal diffusion, the electrode was lowered into place 15 min before the start and was retracted 5–10 min after the end of stimulation. Three to seven days after injections, animals were perfusion fixed (4% PFA) and the brains were processed (see below).

Anterograde Cre-dependent rAAV2-CAG-FLEX-ArchT-GFP (UNC Vector Core, 2.0 × 10¹² titer; n = 5 VGAT^Cre mice and 3 PV^Cre mice) or rAAV8/PAAV-hSyn-DIO-EGFP (UNC Vector Core; 8 × 10¹² titer; n = 1 PV^Cre mouse) were pressure injected (400 nl/mouse) using a glass pipette (5 μl; Harvard Apparatus) with tip diameter of 10–15 µm into the medial septum of mice (stereotaxic coordinates relative to Bregma: 0.86 mm anterior, 0.39 mm lateral and 3.75 mm ventral with 5° angle). Pressure was applied using a 1-μl syringe at a rate of ~ 100 nl/min. To minimise tissue damage and dorsal diffusion, the electrode was lowered into place 5 min before and was retracted 10 min after the injection. After a minimum of 28 days and allowing optimal virus expression, mice were perfusion fixed (2% PFA) and the brains were processed (see below).

Implantation of the head-mounted recording setup, craniotomy and duratomy were performed as previously published (Katona et al. 2017; Lapray et al. 2012). Briefly, rats were anaesthetised (IsoFlo, Abbott), placed into a stereotaxic frame (Kopf Instruments), a micro-drive holder together with a main connector supported by five stainless steel screws was attached to the skull using dental acrylic (Refobacin R, Biomet) and everything was coated with blue light-polymerised cement (Tetric EvoFlow, Ivoclar Vivadent). One of the screws was connected to recording EEG and another for reference-ground signals. After a week, either a single wire electrode (50-μm tungsten, California Fine Wire) fixed on the skull or movable by a miniature drive (Haiss et al. 2010) or a tetrode movable by a microdrive was lowered into the hippocampus or cortex. A layer of silicone (Kwik-Sil, World Precision Instruments) was used to protect the cortical surface overnight and between recordings.

On recording days (1–12 days after duratomy), after short anaesthesia, a miniature preamplifier (ELC mini-preamplifier, NPI Electronic), two LEDs and an accelerometer (Supertech Instruments) were connected to the head stage. A glass electrode (10–20 MΩ) filled with neurobiotin (Vector Laboratories Ltd, 1.5 or 3% w/v in 0.5 M NaCl) was advanced using a hydraulic (Narishige) microdrive to record single cell activity and LFPs in the hippocampus. During recordings, the protective layer of silicone was replaced by paraffin wax (Sigma-Aldrich) for the ease of advancement of the glass electrode. Recordings commenced 1 h after recovery from anaesthesia in a darkened room using a recording arena (50 × 50 cm floor, 27 cm walls) to which the rats were naive on the first day. Two video cameras captured the free movements of the rats; one infra-red sensitive for behavioural analysis and another one for position tracking using the head-mounted LEDs (Position Tracking System, v14.02.08, courtesy of K. Allen). After recording a neuron, the pipette was advanced towards the cell into a juxtacellular position and labelling using neurobiotin was attempted (Pinault 1996). The cell was stimulated by 200 ms of gradually increased positive current pulses to modulate its firing using ELC-03M amplifier with active bridge compensation. When labelling was judged successful due to strong modulation of the cell’s firing, the pipette was retracted slowly and the neuron was left to recover from the entrainment. The rats were then briefly anaesthetised in the stereotaxic frame using isoflurane while the recording equipment was removed. Following a post-labelling period of up to 3 h, the animals were deeply anaesthetised using isoflurane followed by intraperitoneal injection of an overdose of Pentobarbital (JML Biopharm; 20% w/v solution; dose: 400 μl/100 g) and perfusion fixed (4% PFA; see below). If no stable neurons were found, sessions were finished by simply removing the recording devices and by covering the cortical surface with a layer of silicone between consecutive days.

Electrophysiological signals were amplified 1000× (BF-48DGX and DPA-2FS, NPI Electronic) and were digitised at 20 kHz (Power1401 A/D board, Cambridge Electronics Design). Measurements from the glass electrode were online bandpass filtered according to three different frequency ranges (0.3 Hz–10 kHz, wide-band; 0.3–500 Hz, LFP; 0.8–5 kHz, action potentials). Signals from the EEG and hippocampal/cortical electrodes or tetrodes were bandpass filtered (0.3 Hz–10 kHz). Elimination of 50 Hz noise without phase-shift was provided by Hum Bugs (Quest Scientific Instruments). Accelerometer measurements were digitised at 20 kHz. Acquisition of all signals, except video tracking, went in parallel using Spike2 software (v7.06a, Cambridge Electronic Design, ced.co.uk).

Animals were anaesthetised with either sodium pentobarbital (50 mg/kg, i.p.) or chloral hydrate (350 mg/kg) and transcardially perfused with saline followed by either 2% or 4% paraformaldehyde (wt/vol, PFA, Sigma-Aldrich), 15% saturated picric acid (vol/vol, Sigma-Aldrich) and 0.05% glutaraldehyde (wt/vol, distilled grade, TAAB Laboratories Equipment Ltd) in 0.1 M phosphate buffer (PB) at pH 7.2. After brain removal, coronal sections were produced (70 μm nominal thickness, VT1000s vibratome, Leica Instruments). Sections were stored in 0.1 M PB with 0.05% sodium azide at 4 °C.

For immunohistochemistry, indirect primary antibody detection method was used in combination with fluorochrome-conjugated secondary antibodies as described before (Katona et al. 2017; Lasztoczi et al. 2011; Unal et al. 2015). The expression of cell type-specific molecules was tested on either virally transfected individual cells or cells recorded and labelled using neurobiotin and on control tissue. Immunoreactivity was assessed visually by comparing neighbouring cells within the same field of view. A positive signal in the cell of interest was accepted if the subcellular location (e.g. plasma membrane), pattern, and strength of the signal were as expected and similar to those in unlabelled cells. Cells were considered immunonegative for a molecule when fluorescence was undetectable in the tested part of the cell in an area where similar parts of other unlabelled cells were immunopositive. Primary antibodies were used for the following molecules at given dilutions: calbindin (CB, Swant Cat# CB 38, RRID:AB_10000340, 1:500, 1:5000); cannabinoid receptor-1 (CB1, Frontier Institute Cat# CB1-GP, RRID:AB_2571593, 1:1000 and Frontier Institute Cat# CB1-Rb, RRID:AB_2571591, 1:1000); choline acetyltransferase (ChAT, Synaptic Systems Cat# 297 013, RRID:AB_2620040, 1:1000); calretinin (CR, Swant Cat# CG1, RRID:AB_10000342, 1:1000 and Synaptic Systems Cat# 214 102, RRID:AB_2228331, 1:500); enhanced green fluorescent protein (EGFP, gifted by Prof. K. Rockland, RIKEN, rabbit, (Tomioka and Rockland 2006), 1:100, 1:2000 and guinea pig, (Tomioka and Rockland 2007), 1:1000); glutamate decarboxylase (GAD, Millipore Cat# MAB351, RRID:AB_2263126, 1:500); green fluorescent protein (GFP, Molecular Probes Cat# A-11120, RRID:AB_221568, 1:5000 and Aves Labs Cat# GFP-1020, RRID:AB_10000240, 1:1000, 1:10,000); muscarinic acetylcholine receptor M2 (Millipore Cat# MAB367, RRID:AB_94952, 1:250, 1:400, 1:1000); metabotropic glutamate receptor-7a [mGluR7a, gifted by Prof. R. Shigemoto, Division of Cerebral Structure, Nat. Inst. Physiol. Sci, Okazaki, rabbit, (Shigemoto et al. 1996; 1997), 1:500]; metabotropic glutamate receptor-8a [mGluR8a, gifted by Prof. R. Shigemoto, Division of Cerebral Structure, Nat. Inst. Physiol. Sci, Okazaki, guinea pig, (Kinoshita et al. 1996), 1:500, 1:2000 and Santa Cruz Biotechnology Cat# sc-30300, RRID:AB_2116478, 1:800, 1:1000, 1:2000 requiring 2% PFA]; Netrin-G1 (rabbit, 1:1000); Phaseolus vulgaris-leucoagglutinin (PHAL, Vector Laboratories Cat# AS-2224, RRID:AB_2315141, 1:5000 and Vector Laboratories Cat# AS2300, RRID:AB_2315142, 1:500); parvalbumin [PV, Swant Cat# 235 (from 2009), mouse, 1:5000, 1:7500 and Swant Cat# PV27, RRID:AB_2631173, 1:500 and Synaptic Systems Cat# 195 004, RRID:AB_2156476, 1:5000]; somatostatin [SM, gifted by A. Buchan, Medical Research Council Regulatory Peptide Group, Vancouver, British Columbia, mouse, (Vincent et al. 1985), 1:200 and GenWay Biotech Inc. Cat# 18-783-76392-1 ml, RRID:AB_1027453, 1:100]; vesicular acetylcholine transporter (VAchT, Millipore Cat# ABN100, RRID:AB_2630394, 1:3000); vesicular GABA transporter (VGAT, Frontier Institute Cat# VGAT-Rb, RRID:AB_2571622, 1:500 and Synaptic Systems Cat# 131 003, RRID:AB_887869, 1:500 and Frontier Institute Cat# VGAT-Go, RRID:AB_2571623, 1:500 and Synaptic Systems Cat# 131 004, RRID:AB_887873, 1:500); vesicular glutamate transporter-1 (VGLUT1, Synaptic Systems Cat# 135 311, RRID:AB_887880, 1:500); vesicular glutamate transporter-2 (VGLUT2, Synaptic Systems Cat# 135 402, RRID:AB_2187539, 1:500 and Synaptic Systems Cat# 135 404, RRID:AB_887884, 1:1000, 1:2000); vasoactive intestinal polypeptide (VIP, gifted by G. Ohning, Cure, UCLA, mouse, (Wong et al. 1996), 1:30,000, 1:50,000 and ImmunoStar Cat# 20077, RRID:AB_572270, 1:1000). Specificity information for primary antibodies is provided in Salib et al. (2019) and Viney et al. (2018). Secondary antibodies were reported in Katona et al. (2017). Neurobiotin was visualised by streptavidin-conjugated fluorochromes mixed with the solution.

Z-stacks of 8-bit images were taken of M2+ somata and proximal dendrites using laser scanning confocal microscopy and DIC M27 Plan-Apochromat 63×/1.4 NA or alpha Plan-Apochromat 100×/1.46 NA objectives. Assignment of antibodies and respective fluorophores was based on minimising spectral overlap between neighbouring channels and voxel size was optimised for the mGluR8a channel. Counting was carried out after applying a median filter (see below) to reduce noise. Immunolabelled boutons in apposition to M2+ trilaminar cells were counted from individual optical slices. At each level, mGluR8a+ terminals were selected, checked for co-labelling in other channels within the same and adjacent slices and scored for immunoreactivity. Using stereological principles, the tops of varicosities were counted as positive for one or both of the markers. We have sampled: (1) mGluR8a+ boutons on 35 cells from 3 C57BL/6J mice (2% PFA); (2) mGluR8a+ and GAD+ boutons on 13 M2+ cells from 1 rat (4% PFA); (3) mGluR8a+ and VGAT+ boutons on 15 M2+ cells from 3 rats (4% PFA); (4) mGluR8a+ and VGluT1+ boutons on 3 M2+ cells from 3 rats (4% PFA); (5) mGluR8a+ and VAchT+ boutons on 4 M2+ cells from 2 rats (4% PFA); (6) mGluR8a+ , GAD+ and mGluR7a+ boutons on 9 M2+ cells from 3 rats (4% PFA); (7) PHAL+ and/or mGluR8a+ boutons on 4 M2+ cells in 1 section from 1 rat (4% PFA); (8) GFP+ and VGAT+ boutons from 10 sample views in 1 section from 1 VGAT^Cre mouse (2% PFA); (9) GFP+ and/or mGluR8a+ boutons on 10 M2+ cells in 5 sections from 3 VGAT^Cre mice (2% PFA); (10) GFP+ and PV+ somata in 3 sections from 3 PV^Cre mice (2% PFA); (11) GFP+ and PV+ boutons in 3 sections from 2 PV^Cre mice (2% PFA); (12) GFP+ and VGAT+ boutons on 10 M2+ cells in 4 sections from 2 PV^Cre mice (2% PFA); (13) GFP+ boutons on 23 M2+ cells in 7 sections from 2 PV^Cre mice (2% PFA); (14) GFP+ and mGluR8a+ boutons on 5 M2+ cells in 3 sections from 2 PV^Cre mice (2% PFA).

For high-contrast neurobiotin visualisation, we performed avidin–biotin combined horseradish peroxidase (HRP) reactions with 3,3′-diaminobenzidine (DAB) as chromogen and either 0.002% wt/vol hydrogen peroxide (H₂O₂) as substrate, or H₂O₂ generated continuously from glucose by glucose oxidase (Sigma-Aldrich). The detailed methods have been published previously (Lasztoczi et al. 2011). After the DAB reactions, sections were treated with osmium tetroxide (0.1–1%) in 0.1 M PB, followed by graded dehydration in ethanol (70–100%) and propylene oxide. For EM, sections were contrast enhanced by uranyl acetate (1% wt/vol, TAAB, for 35 min) added to 70% ethanol. At the end, all sections were embedded in epoxy resin (Durcupan ACM Fluka, Sigma-Aldrich) and polymerised (2 day at 60 °C).

After blocking of nonspecific antibody binding sites, two methods of indirect primary antibody detection in combination with DAB histochemistry were used to visualise EGFP+ cells. Most sections (including RT25 s59) were incubated in TBS-Tx containing anti-EGFP primary antibody raised in rabbit (2 nights at 4 °C) recognised by biotinylated anti-rabbit secondary antibody raised in donkey (overnight at room temperature) followed by ABC complex in TBS (2 days at 4 °C) and visualised using DAB (0.05%) in combination with nickel–ammonium sulphate (0.5%) in the presence of H₂O₂ (0.001%, for 20 min). Another two sections (RT25 s57, s58) selected for electron microscopy (EM) were processed with freeze–thaw permeabilisation and then were incubated in TBS without detergent containing anti-EGFP and anti-PV primary antibodies (for 40 h at 4 °C) raised in different species. First, biotinylated anti-rabbit secondary antibody and ABC complex were used to detect EGFP in combination with DAB and nickel–ammonium sulphate (black) using the glucose oxidase method for H₂O₂ generation. Visualisation of PV continued with a HRP-conjugated anti-mouse antibody in combination with DAB only (brown) in the presence of H₂O₂. After the DAB reactions, all sections were treated with osmium tetroxide (1% for EM or 0.3% the rest, for 1 h) in 0.1 M PB, followed by graded dehydration and mounting (see above).

The immunohistochemical reactions were first evaluated using wide-field epifluorescence microscopy using either a Leitz DMRB microscope (Leica, Germany) with epifluorescence illumination (Hg arc lamp, HBO, Osram), optimally selected dichroic mirrors and PL Fluotar 20×/0.5 and 40×/0.7 objectives or an AxioImager.Z1 microscope (Carl Zeiss, Germany) with LED illumination and DIC M27 Plan-Apochromat 40×/1.3 oil, 63×/1.4 oil objectives. For documentation and qualitative evaluation of the reactions, images were taken using either a Hamamatsu Photonics ORCA ER CCD camera (C4747-95; Japan) and Openlab v.5.5.0 software (Improvision, UK) or AxioCam HRm CCD camera and AxioVision 2009, Rel.4.8.1 software (Carl Zeiss, Germany), respectively.

Confocal microscopy was performed using the AxioImager.Z1 microscope (Carl Zeiss) equipped with LSM 710 scan head and an additional alpha 20×, 40×, 63 Plan-Apochromat 100×/1.46 oil objective. Image acquisition and analysis was done using ZEN 2008 software, v5.0 (Carl Zeiss). Tracks were switched frame by frame in sequence of increasing excitation wavelengths. Output signals from four subsequent line scans were averaged. Pinhole sizes were adjusted to near 1 Airy Unit (AU) and were slightly varied to produce the same optical slice thickness (typically 0.7 µm) in all channels. Pixel size and numbers were adjusted according to the Nyquist sampling theory. Channel separation was tested by systematic cross-excitation and detection between the channels.

Conventional transmitted light microscopy was performed using either a Leitz Dialux22 microscope (Leica) equipped with NPL Fluotar 10×/0.3, 25×/0.55, 40×/0.78 and PL Apo 100×/1.32 oil objectives or the AxioImager.Z1 microscope (Carl Zeiss) from above. In the case of the former, images were taken using Canon EOS 40D camera controlled by an EOS Canon Utility software v2.9.0.0.

For all imaging modalities, exposure times were individually set for each image depending on the signal strength and the recorded wavelength. Further editing was only done to brightness and contrast levels and was applied for the entire frame in the whole stack and for each channel separately. To reduce noise and enhance contrast levels images were filtered (median algorithm, kernel size X, Y = 3; kernel size Z, channel = 1). When needed, stacks were maximum intensity projected along the Z-axis.

Sections selected from a recorded and neurobiotin-labelled cell (D37r) and an EGFP+ cell (RT25) were processed for serial section electron microscopy (see above). Representative areas of axonal field in the CA1 area were re-embedded and ultrathin serial sections (~ 60 nm) were cut and mounted on single-slot, Pioloform-coated copper grids for conventional transmission electron microscopy (Philips CM100, Gatan UltraScan 1000 CCD camera). We identified synapses as Gray’s type I (often called asymmetrical) and type II (often called symmetrical) based on their fine structure; type I synapses having a thick postsynaptic density, whereas type II synapses are characterised by a thin postsynaptic density (Gray 1959). All labelled boutons cut at the section plane were followed in serial sections to locate synaptic junctions between them and their postsynaptic targets in strata oriens, pyramidale and radiatum. Electron microscopic identification and classification of targeted profiles in the hippocampus was according to established criteria (Gulyás et al. 1999; Megias et al. 2001) and by following postsynaptic dendrites in consecutive sections. Briefly, in stratum radiatum and oriens, pyramidal cells of the CA1 region have spiny dendrites and receive type I synapses exclusively on their dendritic spines, whereas their dendritic shafts are innervated mostly by type II synapses. In contrast, the overwhelming majority of hippocampal interneurons have aspiny or sparsely spiny dendrites, which receive type II and also type I synapses onto their shafts (Takács et al. 2012). In addition, pyramidal cell dendrites have frequent electron-opaque clumps of dense material (50–100 nm) associated with the endoplasmic reticulum. Such dense bodies have not been observed in interneurons. These features were extracted when postsynaptic dendrites were followed in sufficient number of serial sections. If too few sections were available, or the postsynaptic dendrite was a small profile that did not receive any synapse, the parent neuron remained unidentified. Note that this classification criterion may lead to an underestimation of interneuron dendrites and a corresponding overestimation of pyramidal cell dendrites. First, interneuron dendrites may get misclassified as originating from pyramidal cells because of their spines (Takács et al. 2012). Moreover, the density of type I synapses on interneuron dendrites may be low, in which case the short series studied may not reveal such a synapse rendering the shaft unidentified.

The neurobiotin-labelled trilaminar cell (D37r) was digitally reconstructed in 3D and analysed using Neurolucida (MBF Bioscience), on a Nikon Eclipse 80i transmitted light microscope and Lucivid microdisplay (MBF Bioscience) in continuous mode equipped with a VC Plan Apo 100×/1.4 oil immersion objective. Fifty-two coronal sections in consecutive series were reacted with HRP, treated with osmium tetroxide and embedded in resin. Because three sections were lost during processing, neuronal processes were traced from 49 sections and, for the gaps, fragments of axon and dendrite were copied and scaled in X, Y and Z to the right lengths to connect with branches from two neighbouring sections. This extrapolated reconstruction was used for laminar quantification of dendritic and axonal lengths. Individual axonal fragments were split at their crossing points of the laminar boundaries and colour coded accordingly. The pre-spliced lengths were multiplied by the factor 52/49 to estimate the lengths that would have been present in the full cell, had the three sections not been lost.

Tissue shrinkage due to histological processing was corrected (factors reported as mean ± standard deviation) by expanding all elements within individually traced sections in X-, Y-, and Z-dimensions according to criteria we have previously published (Tukker et al. 2013). Briefly, sample sections (n = 31) incubated in 4′,6-diamidino-2-phenylindole (DAPI) were measured for thickness before (i.e. wet) and after (i.e. embedded) either treatment with TBS-TX (n = 3 sections) or F-T (n = 28 sections) using a 63×/1.4 objective and epifluorescence illumination. The thickness (Z) was calculated from the digital readout of a closed-loop z-stage of a motorised microscope (AxioImager.Z1, Carl Zeiss). Shrinkage correction of embedded sections was started by expanding elements within TBS-TX sections in X and Y to the size of those in F-T sections (1.1 ± 0.04 correction factor) enabling the alignment and matching of the processes. Next, the thickness of each embedded section was restored to that before treatment using correction factors (1.4 ± 0.3 for TBS-TX; 1.1 ± 0.1 for F-T) obtained by dividing measured wet thicknesses by those embedded. For TBS-TX sections that had no wet thickness measurements (n = 9), the correction factor (1.4 ± 0.2) was estimated using the mean wet section thickness from all the measured sections (73.0 μm). For F-T sections that had no wet measurements (n = 14), the average factor from the measured F-T sections was used. Finally, all sections were aligned and individually scaled up to wet size in X and Y by applying the published correction factor (1.04) calculated from measurements of sections with the same type of processing (Tukker et al. 2013).

In stratum oriens of both rat and mouse, mGluR8a+ GABAergic boutons are present on somata and proximal dendrites of strongly M2+ neurons in both areas CA1 and CA3 (Figs. 1c–f, i, j, 2a, b). Quantification in the rat revealed that, on average, 89.1% of mGluR8a+ inputs on presumed trilaminar cells are GABAergic and this proportion is similar between neurons in areas CA1 and CA3 (n = 841; χ²(1) = 0.535, p = 0.464; Table 1). In the CA1 area, from a total of 514 mGluR8a+ boutons on presumed trilaminar cells, 89.7% were GABAergic expressing either GAD or VGAT (χ²(1) = 0.570, p = 0.45; Table 1). In the CA3 area, from a total of 327 mGluR8a+ boutons, we identified 88.1% to be GABAergic expressing either GAD or VGAT (χ²(1) = 3.518, p = 0.061; Table 1).

Next, we probed the co-existence of mGluR7a, mGluR8a and GAD in the boutons innervating somata and proximal dendrites of trilaminar cells in the rat (Figs. 2a, b, 5k; Table 2). From a total of 189 mGluR8a+ boutons, 66.1% were GABAergic and mGluR7a+ (Table 2). Of all the GABAergic mGluR8a+ terminals (n = 157, 83.1%), the majority (79.6%) were mGluR7a+ (n = 125; Table 2). Some boutons (8.9%) co-expressed mGluR8a and mGluR7a but were GAD−. Only very few mGluR8a+ terminals (7.9%) were both GAD− and mGluR7a−. Moreover, we encountered rare terminals, which lacked mGluR8a and GAD expression but were mGluR7a+ (Fig. 2a, double arrows).

In the rat, some VIP+ terminals in apposition with trilaminar cells are GABAergic VGAT+ (Fig. 2d). Also, we verified the co-existence of VIP and mGluR8a+, although in only 5% of mGluR8a+boutons (Fig. 6c; see section below and Ferraguti et al. 2005); the majority of the VIP+ terminals on M2+ cells lacked mGluR8a immunoreactivity. Moreover, we found presynaptic boutons on M2+ cells co-expressing PV and VGAT which lacked immunoreactivity for mGluR8a (Fig. 2f).

In the mouse, in addition to terminals expressing VIP and PV (Figs. 2c, e, 5j), we have also observed calbindin-expressing (CB+) and M2+ terminals in apposition to mGluR8a-decorated M2+ cells in stratum oriens; these boutons also lacked immunoreactivity for mGluR8a (data not shown). When tested, terminals strongly expressing the cannabinoid receptor type 1 (CB1) and most likely originating from cholecystokinin-expressing interneurons were also immunonegative for mGluR8a, irrespective of whether they were next to M2+ dendrites or not (data not shown).

Extensive examination of the approximately 10% mGluR8a+ terminals that appeared to lack GABAergic molecular markers and were in apposition to M2+ somata and dendrites in stratum oriens has shown minimal co-localisation between mGluR8a and vesicular glutamate or acetylcholine transporters (see below). We have not tested for the presence of monoaminergic molecular markers in these boutons.

Using immunolocalisation of vesicular acetylcholine transporter (VAchT) in the rat, we have confirmed that some cholinergic terminals in stratum oriens co-express mGluR8a and innervate M2+ cells (Ferraguti and Shigemoto 2006). Furthermore, although very few cholinergic mGluR8a+ terminals are in apposition to each individual M2+ neuron, these were all VGAT+ (n = 12 boutons on 4 cells in 2 rats; see also (Takács et al. 2018)).

In rats, we labelled neurons using Phaseolus vulgaris-leucoagglutinin (PHAL+) in the medial septum (Fig. 3a, b) and their axon collaterals entering and branching in all layers of the hippocampus (Fig. 3e, g, h). In both the CA1 and CA3 areas, PHAL+ terminals targeted somata and dendrites of M2+ hippocampal cells (Fig. 3e, g, h). Each postsynaptic cell was innervated by numerous PHAL+ boutons, and the M2+ cells included those densely decorated by mGluR8a+ terminals (Fig. 3g, h; n = 13 ± 4 terminals, see “Materials and methods”). Some of the PHAL+ septal boutons targeting M2+ cells expressed mGluR8a (Fig. 3h, n = 10 out of 13 terminals; see “Materials and methods”) although many were immunonegative for the receptor. These results reveal the existence of an mGluR8a+ medial septo-hippocampal neuron population, some of which innervate trilaminar cells.

To gain neurotransmitter type specificity, we pursued experiments in mice. First, repeating PHAL labelling of medial septal neurons (Fig. 3c, d) and their axon collaterals in hippocampus (Fig. 3f, i, j), we found that their terminals are VGAT+ and innervate M2+ cells (Fig. 3i, j; 19 out of 23 tested on 3 cells in 1 mouse).

Next, performing viral injections into the medial septum of VGAT^Cre mice (Fig. 4; n = 5 animals; see “Materials and methods”), resulted in the expression of GFP in transfected GABAergic cell bodies, dendrites and axons in medial septum (GFP^VGATCre+; Fig. 4a). As expected, GFP^VGATCre+ medial septal axons were found in all layers of the hippocampus (Fig. 4b–h; 97.0% VGAT+; n = 164 out of 169 terminals; see “Materials and methods”). Focusing on stratum oriens, we confirmed that M2+ neurons co-expressed Netrin-G1 as reported by single-cell and in situ RNA sequencing (Harris et al. 2018; Qian et al. 2019) and demonstrated following transcriptomic profiling of VIP-LRP cells (Luo et al. 2019). Each neuron was innervated, on average, by 12 ± 2 GFP^VGATCre+ medial septal terminals along their cell bodies and proximal dendrites in a single section, in both the CA1 and the CA3 areas (Fig. 4c–f; Table 3; median ± interquartile range; n = 121 terminals). Furthermore, some such cells postsynaptic to GFP^VGATCre+ medial septal axon terminals were densely innervated by 22 ± 8 mGluR8a+ boutons per cell in a single section (n = 10 cells), identifying them as trilaminar cells (Fig. 4e, f; Table 3; median ± interquartile range; n = 237 terminals). Co-expression of mGluR8a was found in 28.9% of the GFP^VGATCre+ medial septal boutons targeting trilaminar cells (Fig. 4f, g; Table 3; n = 35 boutons).

Viral injections into the medial septum of PV^Cre mice (Fig. 5; n = 4 animals; see “Materials and methods”) enabled the expression of GFP in transfected PV+ GABAergic cell bodies, dendrites and axons (GFP^PVCre+; Fig. 5a, b) marking one subpopulation of GABAergic long-range projecting medial septal neurons. PV-immunoreactivity was confirmed in 84.1% of 214 GFP^PVCre+ somata (see “Materials and methods”) with 57.3% transfection rate (n = 180 out of 314 PV-immunoreactive somata; see “Materials and methods”).

In the hippocampus, we observed a high density of virally labelled PV+ septal axons in both areas CA1 and CA3 with a bias towards the latter (Fig. 5c–j; confirming Fig. 8a in Salib et al. 2019). The co-expression of virally delivered GFP and PV was verified in the boutons of these medial septal collaterals (Fig. 5i; 90.7% PV+ ; n = 185 out of 204 terminals; see “Materials and methods”). In another set of experiments, we confirmed that these GFP^PVCre+ septal terminals are GABAergic using VGAT immunoreactivity (Fig. 5j; 98.4% VGAT+; n = 183 out of 186 terminals; see “Materials and methods”).

On average, 15 ± 11 axon terminals of the GFP^PVCre+ medial septal neurons targeted M2+ cells along their cell bodies and proximal dendrites in a single section (Fig. 5e–j; Table 3; median ± interquartile range; n = 356 terminals). Some, but not all, such postsynaptic target neurons (Fig. 5g–j) were also observed being densely decorated by 24 ± 10 mGluR8a+ terminals per cell, identifying them as trilaminar cells (Table 3; median ± interquartile range; n = 137 terminals; see “Materials and methods”). No co-localisation was found in septal terminals between the GFP^PVCre (n = 100) and mGluR8a+ (n = 137; see “Materials and methods”) on these cells.

In summary (Fig. 5k), the very dense mGluR8a+ input of hippocampal trilaminar cells is mostly GABAergic and these terminals often co-express mGluR7a. Much of this GABAergic input originates from medial septal neurons. Specifically, around one-third of the GABAergic medial septal terminals are mGluR8a+; and mGluR8a immunonegative terminals are largely from medial septal PV+ neurons. Presumably, other local hippocampal GABAergic neurons contribute to innervating trilaminar cells with VIP+, PV+, CB+ and M2+ presynaptic terminals, which are predominantly mGluR8a immunonegative.

Following neurobiotin labelling of this trilaminar cell in the dorsal CA1 area, the cell was reconstructed (Fig. 7a, b) through 52 consecutive coronal sections of 70 μm thickness each. After correcting for tissue shrinkage, the complete length of dendrites was 8.3 mm, and the recovered axonal arbour was 62 mm in length including long-range projections innervating the subiculum (see “Materials and methods”).

The cell body was located in stratum oriens close to the border with the alveus and the large horizontal dendritic tree was contained within stratum oriens (Fig. 7a, inset). Expression of M2 in the neurobiotin-labelled somatic and dendritic membranes and a dense innervation by mGluR8a+ boutons (Fig. 7c, d) support the identification of this neuron as a trilaminar cell. The mGluR8a+ presynaptic terminals were mostly GABAergic, as shown by co-localization with either GAD (Fig. 7d bottom) or VGAT, and co-expressed low levels of mGluR7a. Furthermore, from a sample of 79 mGluR8a+ boutons in apposition with the soma and proximal dendrite, 5% (n = 4) were VIP+ (Fig. 7c); the trilaminar cell soma was VIP immunonegative.

The highest proportion of local collaterals was in stratum oriens/alveus and smaller ramifications in strata pyramidale and radiatum with the exclusion of stratum lacunosum moleculare (Fig. 7b, inset). Using serial section electron microscopic quantification of postsynaptic target profiles, we determined that, similar to RT25 above, this trilaminar cell also made synapses preferentially with interneurons (84%, n = 16/19 identified targets; Table 4) and some spiny dendrites, which probably originated from pyramidal cells (Fig. 7e, f). In a small proportion of cases, the identity of the postsynaptic neurons could not be confirmed (Fig. 7e). Often we observed a row of postsynaptic ‘Taxi bodies’ of electron-opaque dots co-aligned with the synaptic junction in the postsynaptic dendrites (Fig. 7f, right; Taxi 1961; Taxi and Babmindra 1972) whose molecular identity remains to be determined. Collaterals of the main axon travelled within stratum oriens or at the border with alveus and the cortical white matter for a distance of 2.8 mm from the soma (40 sections of 70 μm thickness) in the caudo-lateral direction before reaching subiculum where these branched extensively. As subicular terminals could not be visualised due to suboptimal neurobiotin labelling, the identification of postsynaptic target neuron types was not attempted.

During the ~ 9.5-min recording period, the rat was allowed to freely explore and rest in a familiar open field (Fig. 7g, h). The time periods were segmented as movement, quiet wakefulness, slow wave sleep and epochs of rapid eye movement (REM) sleep, facilitating behavioural state-dependent analyses of the trilaminar cell activity (Fig. 8; Table 5).

During slow wave sleep, the mean action potential frequency was 21.6 Hz. The irregular low-frequency spiking of the cell was interspersed with frequent bursts at very high frequencies (> 200 Hz) which often co-occurred with events of high network activation in the LFP resembling 130–230 Hz SWRs. (Fig. 8a–c). During movement, a very different pattern emerged with periodical regular firing at the much lower mean rate of 8.5 Hz, which decreased further to 1.8 Hz during REM sleep (Fig. 8a). The higher firing rate during sleep compared to movement was similar to some identified long-range projecting SST+ cells (Fig. 8c, g; Katona et al. 2017). The minimal activity of the trilaminar cell during REM sleep contrasts with the maximal activation (24.9 Hz) of a recorded long-range projecting SST+ cell (DS17c, Katona et al. 2017). During quiet wakefulness, the mean spike rate of the trilaminar cell (19 Hz) was similar to that during slow wave sleep and to the mean spike rates of all projection cells during movement (14.7 ± 17.7 Hz, median ± interquartile range; Fig. 8c, g and Katona et al. 2017).

To evaluate rhythmicity in the trilaminar cell firing, we calculated autocorrelograms (Fig. 8c; Table 5) and discovered behaviour-dependent variability in the spike trains. We extracted the time points of the first and, if present, the second and third peaks within a 300 ms range. We found an early increase in firing at 3 ms during all states, evidence for the high-frequency bursts of the trilaminar cell as reported under urethane anaesthesia (Ferraguti et al. 2005) and resembling the complex spike bursts fired by pyramidal cells. In contrast to the SST+ projection cells (Katona et al. 2017), additional peaks were found between 105 and 150 ms and 225 and 280 ms reporting 7–10 Hz theta- and 4 Hz delta-rhythmic modulation, respectively.

The median inter spike interval (ISI) lengths across behavioural states were consistently less than 9 ms which is specific to this cell type amongst hippocampal interneurons reported so far (Table 5). We detected behavioural state-dependent differences in the ISI distributions with the interquartile ranges extending between 5.6 ms during sleep–wake transition and 447.2 ms during REM sleep. We monitored the burst firing of the trilaminar cell of three or more action potentials with ISIs shorter than or equal to 12 ms during movement and during slow wave sleep (Fig. 8a, b). Bursts were common throughout the recording and the bursting frequency was 2.5 times higher during slow wave sleep than during movement as also measured by the negative burst frequency index of − 0.43.

During movement, REM sleep and periods of quiet wakefulness, we detected theta frequency (5–12 Hz) rhythmic network activity in the hippocampal LFP (Fig. 8a; Table 5). During theta oscillations, the trilaminar cell fired with a mean rate of 5 Hz—lower than most SST+ projection cells—and ISI lengths of 8.9 ± 114.6 ms (median ± interquartile range)—much shorter than that of SST+ projection neurons. The cell’s firing was strongly phase-coupled to theta cycles with mean vector length of 0.47 (Fig. 8e, f; Table 5).

We have segmented theta epochs according to different behavioural states and have found differences in the distribution of preferential firing rates and phases (Fig. 8d, e; Table 5). There was a striking 67% drop of the mean firing rate during REM sleep compared to other theta periods. Also, the mean preferential theta phases were differently distributed along the ascending slope from 75° during movement to more advanced 130° during sleep–wake transition periods (Fig. 8e; 0° denotes the trough of the extracellularly recorded LFP theta oscillations). The increased firing of the trilaminar cell during the ascending slope is 180° out of phase as compared to the majority of SST+ long-range projection cells firing along the descending slope (Fig. 8f). The trilaminar cell was not significantly coupled to theta cycles during quiet wakefulness.

Action potential autocorrelograms during theta oscillations demonstrate intermittent trilaminar cell activity (Fig. 8a, d; Table 5). Unlike the majority of hippocampal GABAergic neurons, the trilaminar cell did not fire action potentials on every theta cycle (n = 21.8% of all cycles) and when it was active it discharged very few action potentials per cycle. The discontinuous spiking during theta was most pronounced during REM sleep. Interestingly, we also observed a secondary peak in the REM-related autocorrelogram demonstrating an additional slow (1.7 Hz) modulation of the trilaminar cell specifically during this stage of sleep (Fig. 8d). Additionally, in the spike bursts during theta epochs we found three or more action potentials with ISIs shorter than or equal to 12 ms during 6.2% of theta cycles. This is similar to the theta-bursting of three SST+ long-range projecting neurons.

High levels of the inhibitory M2 receptors in the somato-dendritic membranes of trilaminar cells are indicative of their regulation by acetylcholine (Dutar and Nicoll 1988; Seeger and Alzheimer 2001; Tateyama and Kubo 2018; Zheng et al. 2011), but we have not tested any such effects directly. Some interneurons with horizontal dendrites are hyperpolarised by muscarine in stratum oriens (Parra et al. 1998). Furthermore, the non-selective cholinergic agonist carbachol reduces responses to applied glutamate on interneurons in stratum oriens via G-protein activated inwardly rectifying potassium channels, and this modulation is dependent on the expression of M2 (Zheng et al. 2011). Such an inhibitory mechanism is consistent with the low levels of activity of the trilaminar cell we have recorded during theta oscillations associated with exploration or REM sleep, when the cholinergic tone is high in the hippocampus (Kametani and Kawamura 1990; Marrosu et al. 1995). We predict that the cholinergic inhibition of the long-range projecting trilaminar cells contributes to the enhancement of hippocampo-subicular interactions in a network state-dependent manner.

We have established that the majority of the mGluR8a+ terminals on M2+ trilaminar cells are GABAergic and that many originate from the medial septum. Indeed, mRNA for mGluR8a has been shown in a subpopulation of medial septal cells (data from the Allen Brain Atlas) although it remains unknown if any of these neurons would project to the hippocampus.

The medial septal terminals presynaptic to trilaminar cells probably originate from at least two still unrecognised different cell types: (1) because of the lack of mGluR8a in medial septal terminals labelled in PV^Cre mice and innervating trilaminar cells, we conclude that the mGluR8a+ GABAergic medial septal input identified in VGAT^Cre mice originates from PV− cells. Such terminals are similar to those of low-rhythmically firing medial septal cells, which are also PV− (Salib et al. 2019). But whether such cells innervate M2+ neurons in the hippocampus remains to be tested. (2) The numerous PV+ presynaptic terminals making synapses on trilaminar cells would predict a subpopulation of high-rhythmically firing PV+ medial septal cells although these have not been observed to target M2+ hippocampal neurons (Borhegyi et al. 2004; Hangya et al. 2009; Joshi et al. 2017).

Local interneurons in the hippocampus are another likely source of the GABAergic and mGluR8a+ terminals innervating trilaminar cells. This is supported by the sparse distribution of mGluR8a mRNA in the hippocampus interpreted as restricted expression in some rare neuron type and not in pyramidal cells (Ferraguti et al. 2005; Harris et al. 2018). With their inputs proven to express VIP, trilaminar cells are likely candidates for receiving inputs from interneuron selective IS-III cells (Acsády et al. 1996; Chamberland et al. 2010; Lacaille et al. 1987; Tyan et al. 2014) immunopositive for both VIP and CR, or from other CR immunonegative VIP+ neurons including VIP-LRPs co-expressing M2 (Acsády et al. 1996; Francavilla et al. 2018; Gulyás et al. 1996; Hájos et al. 1996; Turi et al. 2019). Boutons of the VIP+ IS-III cells express high levels of mGluR7a (Cauli et al. 2000; Dalezios et al. 2002) and we found a high proportion of mGluR7a co-expression in the mGluR8a+ GABAergic terminals innervating trilaminar cells. We confirmed the co-localisation of mGluR8a and VIP in some (as in Ferraguti et al. 2005), but were unable to test the terminals for all three markers. Whether the VIP+ input originated from any of the IS cells remains to be established. In addition, it remains to be determined whether some of the mGluR7a expression in GABAergic terminals (Kinoshita et al. 1998; Somogyi et al. 2003) originates from PV+ local interneurons (Cauli et al. 2000; Dalezios et al. 2002; Somogyi et al. 2003).

Medial septal cholinergic fibres are a major neuromodulatory subcortical pathway in the hippocampus (Gielow and Zaborszky 2017; Lewis et al. 1967; Nyíri et al. 2005). Basal forebrain cholinergic terminals may also use GABA as transmitter and form synapses with interneuron dendrites in the hippocampal CA1 (Takács et al. 2018). In the rat, some cholinergic terminals were identified co-expressing mGluR8a and targeting M2+ cells (Ferraguti and Shigemoto 2006). In our samples, we confirmed the co-expression of VAchT and mGluR8a. Although very few such terminals were detected on single M2+ neurons, these terminals were also immunopositive for VGAT. The physiological roles of a co-modulation of trilaminar cells by GABA and acetylcholine remain to be established.

Some mGluR8a+ boutons are non-GABAergic and could be glutamatergic. Terminals of hippocampal CA1 pyramidal cells as examined so far lacked mGluR8a expression (Ferraguti et al. 2005) suggesting that the glutamatergic terminals containing these receptors are likely to originate from extrahippocampal sources. There is no evidence that trilaminar cells receive medial septal glutamatergic inputs (Fuhrmann et al. 2015; Justus et al. 2017; Sotty et al. 2003). Glutamatergic medial septal neurons mostly innervate stratum oriens SST+ neurons (Fuhrmann et al. 2015; Justus et al. 2017). Whether these cells are the only targets and whether these glutamatergic axons co-express mGluR8a needs to be addressed in future experiments. Glutamatergic neurons of the amygdala express mGluR8a (Palazzo et al. 2011) and are another candidate to consider as innervating the hippocampus and trilaminar cells (Pikkarainen et al. 1999), but it is unknown if these projections overlap with the somato-dendritic fields of trilaminar cells. There is a marked expression of mGluR8a in the terminal zones of the lateral perforant path originating from principal cells in the entorhinal cortex (Shigemoto et al. 1997). Furthermore, co-localization of mGluR7a and mGluR8a has been shown in the same glutamatergic terminals (Shigemoto et al. 1997). In our current analysis, some of the mGluR7a+/mGluR8a+ input terminals innervating trilaminar cells were immunonegative for GAD and may be glutamatergic. One group of the entorhinal afferents projects to stratum oriens/alveus and shows a higher preference for innervating GABAergic cells (Takács et al. 2012). Several stratum oriens/alveus SST− interneurons were seen decorated by terminals labelled for mGluR7a and mGluR8a (Ferraguti et al. 2005); in some cases, the labelling for the two receptors appeared co-localised in the same terminal (Ferraguti et al. 2005).

We consider the exclusively mGluR7a+ input terminals innervating trilaminar cells as likely originating from the local axon collaterals of CA1 pyramidal neurons (Bradley et al. 1996; Shigemoto et al. 1996). Recurrent collaterals of CA1 pyramidal cells are a common source of glutamate in stratum oriens but their postsynaptic action is differentiated largely due to the selective enrichment of mGluR7a expression in a target cell-dependent manner, i.e. higher levels are distributed to the terminals that form synapses with SST+ O-LM neurons than to terminals making synapses with pyramidal cells or other types of GABAergic neuron including trilaminar cells (Ali and Thomson 1998; Blasco-Ibáñez and Freund 1995; Losonczy et al. 2002; McBain et al. 1994; Shigemoto et al. 1997, 1996; Stachniak et al. 2019).

Although implicated in the pathophysiology of stress-induced disorders including depression and anxiety (Bahi 2017; Fendt et al. 2010; Niswender and Conn 2010), the precise functional roles of mGluR8a remain to be dissected according to cell types and brain areas where the receptor is expressed (Corti et al. 1998; Duvoisin et al. 1995; Ferraguti 2018; O'Connor et al. 2013; Palazzo et al. 2011; Shigemoto et al. 1997). In glutamatergic boutons, mGluR8a may act as autoreceptor activated by glutamate released from the same terminal (Baskys and Malenka 1991; Desai et al. 1994; Gereau and Conn 1995; Scanziani et al. 1998; Shigemoto et al. 1997; Trombley and Westbrook 1992). The presence of mGluR8a in GABAergic terminals indicates heterosynaptic regulation of GABA release (Desai et al. 1994; Gereau and Conn 1995; Hayashi et al. 1993; Kogo et al. 2004; Morishita et al. 1998; Poncer and Miles 1995; Semyanov and Kullmann 2000).

The presynaptic expression of high-affinity mGluR8a in GABAergic boutons innervating trilaminar cells predicts a role in adjusting the inhibition of these neurons depending on the level of glutamatergic activity in the local network. We propose that the intermittent high population synchrony of pyramidal cell action potentials during SWRs results in synaptically released glutamate spill-over (Congar et al. 1997; Scanziani et al. 1997) and suppression of GABA release to trilaminar cells (Capogna 2004; Dammann et al. 2018; Mitchell and Silver 2000; Scanziani et al. 1998; Semyanov and Kullmann 2000; Takahashi et al. 1996; Woodhall et al. 2001), thereby facilitating their prolonged high-frequency burst firing. This allows trilaminar cells to coordinate principal cell activity between the hippocampus and the subiculum during offline processing.

The repeated high-frequency bursts of firing reported under urethane anaesthesia (Ferraguti et al. 2005; Sik et al. 1995) are a conspicuous feature of the trilaminar cell type that may serve to support CA1 principal cells by disinhibition via transient inhibition of certain types of local interneuron. We suggest that this disinhibition facilitates the activation of pyramidal cells into sequences of cell assemblies replayed during SWRs (Diba and Buzsaki 2007; Foster and Wilson 2006). Alternatively, trilaminar cells may entrain specialised GABAergic interneuron types to ripple frequencies which in turn are responsible for the ripple-rhythmic (120–230 Hz) entrainment of pyramidal cells (Csicsvari et al. 1999; Katona et al. 2014, 2017; Lapray et al. 2012; Varga et al. 2012, 2014) thereby initiating their functional coupling by synaptic plasticity mechanisms (Behr et al. 2009; Buzsáki 1989; Commins et al. 1998; Csicsvari et al. 1999; Kokaia 2000; Wilson and McNaughton 1994). An outstanding question remains whether trilaminar cells via their long-range GABAergic axons also transiently inhibit interneurons in the subiculum and therefore facilitate the co-activation of hippocampal and subicular pyramidal cells promoting the oscillatory coherence between these two brain regions (Böhm et al. 2015; Buzsáki 1986, 1989; Chrobak and Buzsaki 1994; Chrobak and Buzsáki 1996; Eller et al. 2015; Norimoto et al. 2013).

Long-range GABAergic, cholinergic, and glutamatergic projections from the medial septum and diagonal band nuclei (Borhegyi et al. 2004; Colom et al. 2005; Freund and Antal 1988; Hajszan et al. 2004; Joshi et al. 2017; Kimura et al. 1980; Kunitake et al. 2004; Petsche et al. 1962; Unal et al. 2018, 2015; Viney et al. 2018) contribute to the rhythmic entrainment of hippocampal and subicular neurons and the generation of cortical theta oscillations (Brucke et al. 1959; Ford et al. 1989; Hangya et al. 2009; Simon et al. 2006; Sweeney et al. 1992; Varga et al. 2008; Vertes and Kocsis 1997; Wang 2002). Medial septal GABAergic cells have been shown to target exclusively interneurons in the hippocampus (Freund and Antal 1988; Freund and Buzsáki 1996; Joshi et al. 2017; Köhler et al. 1984; Salib et al. 2019; Unal et al. 2018, 2015). We found that trilaminar cells are one of the densely innervated cell types by the GABAergic component of the medial septum, but the theta-related suppression of their activity is markedly different from that of many other identified hippocampal GABAergic neuron type (Joshi et al. 2017; Somogyi et al. 2014). An increase in cholinergic tone, which accompanies theta oscillations, is very likely to contribute to their strong inhibition via M2 receptors (Kametani and Kawamura 1990; Marrosu et al. 1995), as well as, GABAergic gating by VIP+ interneurons. The remarkable decrease in the trilaminar cell firing during theta oscillations may relieve their postsynaptic target interneurons from inhibition. As a result, these interneurons, can orchestrate the theta phase-dependent recruitment of pyramidal cells into temporally ordered local and cross-regional assemblies (Buzsáki 1989, 2010; Commins et al. 1999; Harris et al. 2003; Huang and Kandel 2005; Kokaia 2000; O'Mara et al. 2000; Pastalkova et al. 2008; Somogyi 2010; Wilson and McNaughton 1993, 1994).