Target cell-specific plasticity rules of NMDA receptor-mediated synaptic transmission in the hippocampus

fncel-17-1068472 March 30, 2023 Time: 15:35 # 1 TYPE Original Research PUBLISHED 05 April 2023 DOI 10.3389/fncel.2023.1068472 OPEN ACCESS EDITED BY Dominique Debanne, INSERM U1072 Neurobiologie des Canaux Ioniques et de la Synapse, France REVIEWED BY Diasynou Fioravante, University of California, Davis, United States Hélène Marie, Centre National de la Recherche Scientifique (CNRS), France *CORRESPONDENCE Pablo E. Castillo [email protected] †PRESENT ADDRESS Karina Alviña, Department of Neuroscience, University of Florida, Gainesville, FL, United States ‡These authors have contributed equally to this work SPECIALTY SECTION This article was submitted to Cellular Neurophysiology, a section of the journal Frontiers in Cellular Neuroscience RECEIVED 13 October 2022 ACCEPTED 20 March 2023 PUBLISHED 05 April 2023 CITATION Lutzu S, Alviña K, Puente N, Grandes P and Castillo PE (2023) Target cell-specific plasticity rules of NMDA receptor-mediated synaptic transmission in the hippocampus. Front. Cell. Neurosci. 17:1068472. doi: 10.3389/fncel.2023.1068472 COPYRIGHT © 2023 Lutzu, Alviña, Puente, Grandes and Castillo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Target cell-specific plasticity rules of NMDA receptor-mediated synaptic transmission in the hippocampus Stefano Lutzu1‡, Karina Alviña1†‡, Nagore Puente2,3, Pedro Grandes2,3 and Pablo E. Castillo1,4* 1Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY, United States, 2Department of Neurosciences, Faculty of Medicine and Nursing, University of the Basque Country UPV/EHU, Leioa, Spain, 3Achucarro Basque Center for Neuroscience, Science Park of the University of the Basque Country UPV/EHU, Leioa, Spain, 4Department of Psychiatry and Behavioral Sciences, Albert Einstein College of Medicine, Bronx, NY, United States Long-term potentiation and depression of NMDA receptor-mediated synaptic transmission (NMDAR LTP/LTD) can significantly impact synapse function and information transfer in several brain areas. However, the mechanisms that determine the direction of NMDAR plasticity are poorly understood. Here, using physiologically relevant patterns of presynaptic and postsynaptic burst activities, whole-cell patch clamp recordings, 2-photon laser calcium imaging in acute rat hippocampal slices and immunoelectron microscopy, we tested whether distinct calcium dynamics and group I metabotropic glutamate receptor (I-mGluR) subtypes control the sign of NMDAR plasticity. We found that postsynaptic calcium transients (CaTs) in response to hippocampal MF stimulation were significantly larger during the induction of NMDAR-LTP compared to NMDARLTD at the MF-to-CA3 pyramidal cell (MF-CA3) synapse. This difference was abolished by pharmacological blockade of mGluR5 and was significantly reduced by depletion of intracellular calcium stores, whereas blocking mGluR1 had no effect on these CaTs. In addition, we discovered that MF to hilar mossy cell (MFMC) synapses, which share several structural and functional commonalities with MF-CA3 synapses, also undergoes NMDAR plasticity. To our surprise, however, we found that the postsynaptic distribution of I-mGluR subtypes at these two synapses differ, and the same induction protocol that induces NMDAR-LTD at MF-CA3 synapses, only triggered NMDAR-LTP at MF-MC synapses, despite a comparable calcium dynamics. Thus, postsynaptic calcium dynamics alone cannot predict the sign of NMDAR plasticity, indicating that both postsynaptic calcium rise and the relative contribution of I-mGluR subtypes likely determine the learning rules of NMDAR plasticity. KEYWORDS CA3, dentate gyrus, mossy cell, synaptic plasticity, calcium signal, two-photon laser scanning microscopy Frontiers in Cellular Neuroscience 01 frontiersin.org fncel-17-1068472 March 30, 2023 Time: 15:35 # 2 Lutzu et al. 10.3389/fncel.2023.1068472 Introduction Long-term changes in synaptic strength are widely regarded as cellular correlates of most forms of learning and memory (Malenka and Bear,2004;Mayford et al.,2012). The N-methylD-aspartate receptor (NMDAR) is a critical trigger of long-term potentiation and depression (LTP and LTD) of the fast component of glutamatergic transmission, which is mainly mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) (Luscher and Malenka,2012). Like AMPARs, NMDARs are also dynamically regulated and undergo activity-dependent bidirectional long-term plasticity (i.e., NMDAR-LTP and LTD) at many central synapses (Rebola et al.,2010;Hunt and Castillo,2012). NMDAR plasticity can strongly impact synaptic transmission by changing the threshold of AMPAR plasticity (Rebola et al.,2011;Hunt et al.,2013) and by modulating synaptic integration and neuronal output (Hunt et al.,2013;Banks et al., 2015). However, the molecular mechanisms that control NMDAR plasticity and determine its direction remain unclear. A better understanding of these mechanisms is important since NMDAR dysregulation has been implicated in several brain disorders (Lau and Zukin,2007), including schizophrenia (Javitt,2010), autism spectrum disorder (Won et al.,2012), addiction (Borgland et al., 2006) and neurodegenerative diseases such as Alzheimer’s (Liu et al.,2019) and Parkinson’s disease (Gardoni et al.,2010). The induction of NMDAR plasticity relies on postsynaptic calcium rise, which typically arises from the activation of NMDARs themselves and the recruitment of other calcium sources such as internal calcium stores and calcium influx via voltage-gated calcium channels (Kwon and Castillo,2008a;Rebola et al.,2008; Harnett et al.,2009;Fernandez de Sevilla and Buno,2010;Jo et al., 2010;Hunt et al.,2013). The importance of calcium signaling in the bidirectionality of NMDAR plasticity is highlighted by the fact that changing the intracellular calcium buffer capacity with calcium chelators can either block or change the sign of NMDAR plasticity (Harney et al.,2006). In addition, several G protein-coupled receptors (GPCRs) have been implicated in the induction of NMDAR plasticity (MacDonald et al.,2007;Lutzu and Castillo,2020). Chief among them is the group I metabotropic glutamate receptors (I-mGluRs), which play an essential role in the induction of NMDAR plasticity at several central synapses (Yang et al.,2014;Lutzu and Castillo,2020). I-mGluRs are Gqcoupled protein receptors whose activation induces calcium release from internal stores (Niswender and Conn,2010), indicating that mGluRs significantly contribute to the postsynaptic calcium rise that triggers NMDAR plasticity. At the hippocampal mossy fiberto-CA3 pyramidal cells (MF-CA3) synapse, bidirectional NMDAR plasticity can be elicited by pairing physiologically relevant patterns of presynaptic and postsynaptic burst firing (i.e., burst timing-dependent plasticity), where the order in which preand postsynaptic bursts are presented is crucial to establish the direction of NMDAR plasticity (Hunt et al.,2013). Specifically, NMDAR-LTP is triggered if presynaptic burst firing precedes postsynaptic firing, while simply reversing the order of the bursts induces NMDARLTD. Both NMDAR-LTP and LTD rely on postsynaptic calcium rise but display different molecular requirements. Whereas NMDARLTP requires NMDAR/mGluR5 co-activation and calcium release from intracellular stores, NMDAR-LTD requires NMDAR/mGluR1 co-activation but not calcium release from internal stores (Hunt et al.,2013), suggesting that higher levels of postsynaptic calcium determine the direction of NMDAR plasticity, a possibility never directly tested. Moreover, what additional factors contribute to inducing LTP or LTD of NMDAR-mediated transmission remain unclear. In the present study, we combined whole-cell patch clamp recordings, 2-photon (2P) calcium imaging and glutamate uncaging in acute rat hippocampal slices, and anatomical analysis via immunoelectron microscopy, to determine the role of postsynaptic calcium rise and I-mGluRs in shaping bidirectional NMDAR plasticity. In addition to the MF-CA3 synapse, we also examined the synapse between MF and hilar mossy cells (MF-MC synapse), which shares several anatomical and functional properties with MF-CA3 synapses (Scharfman,2016). Our data indicate that NMDAR plasticity is target-cell specific, and that expression of I-mGluRs subtypes combined with postsynaptic calcium dynamics determine the expression and direction of NMDAR plasticity at MF synapses. Materials and methods All experimental procedures described were performed in accordance with the Institutional Animal Care and Use Committee guidelines of the Albert Einstein College of Medicine and the University of the Basque Country and adhered to the guidelines of the USA National Institutes of Health (NIH). Acute transverse slices were prepared from young adult Sprague Dawley rats of both sexes (P17-27). Hippocampi were dissected from the brain and 400 µm thick slices were cut using a vibratome (Leica VT1200S) in ice-cold sucrose solution containing (in mM): 215 sucrose, 20 glucose, 26 NaHCO3, 4 MgCl2, 4 MgSO4, 1.6 NaH2PO4, 2.5 KCl, and 1 CaCl2. Slices were then allowed to recover for ∼20 min at 34◦C in 50% sucrose solution and 50% artificial cerebral spinal fluid (ACSF) recording solution containing (in mM): 124 NaCl, 26 NaHCO3, 10 glucose, 2.5 KCl, 1 NaH2PO4, 2.5 CaCl2, and 1.3 MgSO4. After this recovery period, slices were incubated in ACSF at room temperature (∼25◦C) for at least one hour. For electrophysiology and calcium imaging experiments slices were perfused with ACSF bubbled with 95% O2and 5% CO2. Unless otherwise stated, during calcium imaging experiments ACSF was supplemented with the GABAAreceptor antagonist picrotoxin (30 µM) to block fast synaptic inhibition, and 0.5–1 µM LY303070, a non-competitive selective AMPAR antagonist, to minimize the activation of the CA3 recurrent network (Kwon and Castillo, 2008b). Electrophysiology Slices were transferred to a recording chamber perfused with ACSF bubbled with 95% O2and 5% CO2and kept at room temperature (∼25◦C). The flow rate was adjusted to 2 ml/min. Visualized whole-cell patch clamp recordings from CA3 pyramidal neurons and MCs were performed using borosilicate glass pipettes (3–6 Mtip resistance) filled with a potassium-based internal Frontiers in Cellular Neuroscience 02 frontiersin.org fncel-17-1068472 March 30, 2023 Time: 15:35 # 3 Lutzu et al. 10.3389/fncel.2023.1068472 solution containing: 135 mM KMeSO3, 10 mM HEPES, 4 mM MgCl2, 4 mM Na2ATP, 0.4 mM NaGTP and 10 mM Na+creatine phosphate; 290 mOsm; pH 7.3 corrected with KOH. For calcium imaging, the low-affinity calcium indicator Fluo5f (300 µM) and the morphological fluorescent indicator Alexa 594 (20 µM) were added to the internal solution. Data were collected using a MultiClamp 700B amplifier (Molecular Devices, San Jose, CA). Signals were filtered at 2.4 kHz and acquired at 5 kHz with custom software written in IgorPro. CA3 pyramidal cells and MCs were initially voltage clamped at ∼–65 mV and the internal solution was allowed to equilibrate for at least 30 min. During this time window, we checked for the presence and stability of MF-derived AMPAR excitatory postsynaptic currents (EPSCs) using an extracellular bipolar theta glass stimulator which was placed in the stratum lucidum for MF-CA3 recordings, or in the subgranular zone of the dentate gyrus for MF-MC recordings. In both cases, the stimulating pipette was placed ∼50–100 µm from the patched cell. MFevoked responses were elicited with a short (80 µs) current square pulse. Stimulation intensity ranged from 20 to 50 µA. For both types of recordings, MF-derived responses were identified according to the high presynaptic facilitation (P2/P1 >2), fast rise time (<2 ms) and high sensitivity to the mGluR2/3 agonist DCG-IV (1 µM) (>80% reduction of the synaptic response). After the initial ∼30 min in voltage clamp, recordings were switched to current clamp mode while keeping the membrane resting potential at −70 mV. The resting potential was adjusted to more hyperpolarized potentials (∼–75 mV) if synapticallydriven spikes were evoked during MF burst stimulation. The induction protocols for NMDAR-LTP and LTD were designed as previously described (Hunt et al.,2013). Briefly, NMDAR-LTP and LTD induction protocols consisted of pairings of presynaptic MF bursts (PRE) (5 pulses at 50 Hz) with postsynaptic bursts of CA3 pyramidal cells (POST) (3 action potentials at 100 Hz) which were evoked with brief (2–3 ms) injections of current (1–1.5 nA) in the postsynaptic cell. The NMDAR-LTP protocol consisted of 100 PRE-POST pairings (i.e., PRE leading POST stimulation) with a 10 ms delay in between, delivered at a 2 Hz inter-pairing interval. NMDAR-LTD was induced by reversing the burst order (i.e., POST-PRE) with the same 10 ms delay. All calcium measurements were performed using single pairings except those in Figures 1G–I, where the full induction protocol with one hundred pairings was used, and in Figure 3 where only PRE stimulation patterns were used. Recordings showing access resistance >25 Mor changes exceeding 15% were discarded. To measure DHPG-induced currents we patched CA1 pyramidal cells in voltage-clamp mode at −60 mV in presence of 10 µM NBQX, 100 µM picrotoxin, and 3 µM CGP and 25 µM D-APV (to block AMPA/Kainate, GABAA, GABAB, and NMDA receptors, respectively). The DHPG-mediated change in holding current was measured by subtracting the average value of the holding current during the last 3 min of a 10-min DHPG application to the average value of the baseline period (1I Holding). For the induction of NMDAR plasticity in MCs, visualized whole-cell patch clamp recordings from MCs were performed using a K+-based internal solution containing: 135 mM KMeSO4, 5 mM KCl, 1 mM CaCl2, 5 mM NaOH, 10 mM HEPES, 5 mM MgATP, 0.4 mM Na3GTP, 5 mM EGTA, 10 mM D-glucose, pH 7.2 (280–290 mOsm). MCs were identified using previously established criteria (Larimer and Strowbridge,2008): elevated spontaneous synaptic activity, little to no afterhyperpolarization and non-burst firing patterns. These criteria were validated by patch-loading cells with Alexa 594, which allowed us to image the cell and confirm the presence of TEs on MCs (Scharfman,2016). To enhance response detectability, MF-derived NMDAR EPSCs were elicited with 2 pulses at 200 Hz in the presence of 10 µM NBQX, 100 µM picrotoxin, and 3 µM CGP (to block AMPA/Kainate, GABAA, and GABABreceptors respectively). 2-photon calcium imaging and glutamate uncaging Calcium imaging and glutamate uncaging measurements were performed using an Ultima 2-photon laser scanning microscope (Bruker Corporation, Billerica, MA) equipped with both an Insight Ti:Sapphire laser and a MaiTai laser (Spectra Physics, MKS Instruments, Inc., Andover, MA). The excitation wavelength for imaging and uncaging were set at 830 and 720 nm, respectively. For calcium imaging, fluorescence across the region of interest was detected in line scan mode at high frequency (500 Hz) using Prairie View 5.4 software (Bruker Corporation). Postsynaptic CaTs were estimated by calculating the 1G/R ratio, which represents the stimulation-induced change in fluorescence from the baseline of the green channel (Fluo-5f signal), normalized by the fluorescence of the Red channel (Alexa-594 signal). PRE, POST, PRE-POST, and POST-PRE CaTs were quantified as the average peak 1G/R of at least 5 trials. All trials were acquired with at least 1 min delay. To assess the effect of drug application on PRE CaTs, the peak 1G/R after drugs wash-in (average of at least 5 traces acquired during a 5–10 min long baseline) was normalized to the average of the CaTs acquired during the baseline period (average of at least 5 traces acquired during the last 15 min of 30 min drug wash-in). Only trials in which synaptic MF stimulation did not induce spikes were analyzed. To avoid potential priming effects due to the PRE-POST, or POST-PRE stimulation, we delivered these protocols in an interleaved fashion in different cells. For glutamate uncaging experiments 2.5 mM MNI-cagedL-glutamate was added to the ACSF and used to perfuse the slice. The uncaging laser was parked ∼1µm away from the head of the target dendritic spine and 5 uncaging pulses (0.5 ms duration, at 50 Hz) were used to mimic glutamate release from MFs with the same frequency used for electrical stimulation of MF. The uncaging laser power was adjusted to detect a measurable calcium signal and avoid postsynaptic spikes during the short burst. To compare the difference between 100 PREPOST and POST-PRE pairings, measurements were performed in the same cell. To avoid any potential confounding effect due to changes in NMDAR function, the two protocols were elicited within 5 min from each other, before the onset of the expression of NMDAR plasticity (Hunt et al.,2013). Moreover, the two protocols were elicited by alternating the order in different cells to account for potential priming effects between protocols. Frontiers in Cellular Neuroscience 03 frontiersin.org fncel-17-1068472 March 30, 2023 Time: 15:35 # 4 Lutzu et al. 10.3389/fncel.2023.1068472 FIGURE 1 NMDAR LTP was associated with a larger postsynaptic calcium rise than NMDAR LTD at the MF-CA3 synapse. (A) Experimental recording configuration (top). Simultaneous excitation of Alexa 594 and Fluo-5f loaded via a patch pipette was performed with a 2P laser tuned at 830 nm. Representative CA3 pyramidal cell loaded with Alexa 594 (middle) and inset showing magnification of the imaged postsynaptic TE (yellow box). High magnification image showing the scanning line (yellow dashed line) on the TE (bottom). s.l.: stratum lucidum; θstim: theta glass stimulus pipette; rec: recording pipette. (B) Electrophysiological recordings and calcium responses obtained from a CA3 pyramidal neuron. Stimulation patterns employed (top): PRE (MF electrical stimulation – 5 pulses at 50 Hz); POST (CA3 pyramidal cell action potentials – 3 spikes at 100 Hz); PRE-POST (delay 10 ms between bursts); and POST-PRE (delay 10 ms between bursts). Fluorescence profile (middle) of the green signal (Fluo5f) superimposed with the red signal (morphological dye Alexa 594). S: spine; d: dendrite. Fluorescence quantification reported as 1G/R over time (bottom). (C) Summary of mean peak 1G/R across all patterns of stimulation. Here and in all figures, circles linked by a line represent individual experiments performed in the same cell. (D) Experimental configuration used to perform electrophysiology, calcium imaging and glutamate uncaging (top). Excitation wavelengths employed were 830 nm (for imaging) and 720 nm (for glutamate uncaging). High magnification image showing the position of the uncaging laser beam (yellow circle) the scanning line (yellow dashed line) on the TE (bottom). (E,F) Same as panels (B,C) but employing glutamate uncaging instead of MF stimulation. (G) Single electrophysiological response (left) and fluorescence profile during full induction protocol for NMDAR LTP and LTD (right). (H) Calcium responses of the experiment showed in panel (G) expressed as 1G/R over time. Boxed area shows a magnification of 1G/R during the first 4 pairings. (I) Summary of 1G/R change during the full induction protocols expressed as integrated area under the curve. Data are presented as mean ±SEM. **p<0.01, ***p<0.001. Double pre-embedding immunoelectron microscopy Long Evans rats (n= 3) were deeply anesthetized by intraperitoneal injection of ketamine/xylazine (80/10 mg/kg body weight) and transcardially perfused at room temperature (RT, 20– 25◦C) with phosphate buffered saline (0.1 M PBS, pH 7.4) for 20 s, followed by the fixative solution (4% formaldehyde freshly depolymerized from paraformaldehyde, 0.2% picric acid and 0.1% glutaraldehyde) in PBS (0.1 M, pH 7.4) for 10–15 min. Brains were removed from the skull and post-fixed in the fixative solution for about 1 week at 4◦C and stored at 4◦C in 1:10 diluted fixative solution until use. The procedure has been previously described (Puente et al., 2019). Briefly, coronal hippocampal vibrosections were cut at 50 µm and collected in phosphate buffer (0.1 M PB, pH 7.4) Frontiers in Cellular Neuroscience 04 frontiersin.org fncel-17-1068472 March 30, 2023 Time: 15:35 # 5 Lutzu et al. 10.3389/fncel.2023.1068472 FIGURE 2 mGluR5 but not mGluR1 activation was critical for the difference in postsynaptic calcium rise during PRE-POST and POST-PRE at MF-CA3 synapses. (A) Representative traces of PRE-POST and POST-PRE fluorescence profiles in control conditions (top) and summary plots showing the average 1G/R measured during PRE-POST and POST-PRE patterns (same dataset from Figure 1C) (bottom). (B,C) Same experimental design as in A but in presence of the mGluR5 antagonist MPEP (4 µM) (B) and the mGluR1 antagonist YM (1 µM) (C).(D) Representative inward current mediated by bath application of the group I mGluR agonist DHPG (30 µM) in control and during YM co-application (top). Time-course summary plot of the DHPG-mediated change in holding current (1I Holding) recorded from voltage-clamped CA3 pyramidal cells under control conditions and in the presence of YM. Here and in all figures: a, number of animals and c, number of cells. Data are presented as mean ±SEM). **p<0.01, ***p<0.001, n.s. not significant. with 0.1% sodium azide at RT. They were transferred and preincubated in a blocking solution of 10% bovine serum albumin (BSA), 0.1% sodium azide and 0.02% saponin prepared in Trishydrogen chloride buffered saline 13 (TBS), pH 7.4 for 30 min at RT. Then, the hippocampal sections were incubated with the primary rabbit polyclonal anti-mGluR1b receptor antibody (2 mg/ml, mGluR1b–Rb–Af250, Frontier Institute Co. Ltd, Ishikari, Hokkaido, Japan, RRID:AB_2616586) or guinea pig polyclonal anti-mGluR5 antibody (2 mg/ml, mGluR5b–GP–Af270–1, Frontier Institute Co. Ltd, Ishikari, Hokkaido, Japan, RRID:AB_2571804) diluted in 10% BSA/TBS containing 0.1% sodium azide and 0.004% saponin on a shaker for 2 days at 4◦C. Both antibodies were extensively characterized previously, and the characteristic labeling in wildtype tissue disappeared in the brains of mGluR1 and mGluR5 knock–out mice (Ferraguti et al.,1998;Mateos et al., 1998;Tanaka et al.,2000;Uchigashima et al.,2007;Ohtani et al., 2014). Tissue was incubated after several washes in 1% BSA/TBS with the biotinylated secondary antibody (1:200 goat biotinylated anti-rabbit, Cat# BA-1000, Vector Labs, Burlingame, CA, USA; RRID:AB_2313606 or 1:200 donkey biotinylated anti-guinea pig Cat#, 706-065-148, Jackson ImmunoResearch Labs, West Grove, PA, USA; RRID:AB_2340451) in 1% BSA/TBS with 0.004% saponin for 3 h at RT. The sections were then washed in 1% BSA/TBS overnight on a shaker at 4◦C and incubated with either 1.4 nm gold-labeled goat anti-rabbit IgG antibody (Fab’ fragment, 1:100, Cat # #2004, Nanoprobes Inc., Yaphank, NY, USA) or 1.4 nm gold-labeled goat anti-guinea pig IgG antibody (Fab’ fragment, 1:100, Cat # #2055, Nanoprobes Inc., Yaphank, NY, USA) in 1% BSA/TBS with 0.004% saponin on a shaker for 3 h at RT, for mGluR1b or mGluR5 labeling, respectively. Next, the sections were washed in 1% BSA/TBS and subsequently incubated in the avidinbiotin complex (1:50; PK-7100, Vector Labs, Burlingame, CA, USA; RRID:AB_2336827) diluted in the wash solution for 1.5 h. After washing in 1% BSA/TBS overnight at 4◦C, tissue was post-fixed with 1% glutaraldehyde in TBS for 10 min and washed in doubledistilled water. Afterward, the gold particles were silver intensified with an HQ Silver kit (Cat#2012; Nanoprobes Inc., Yaphank, NY, USA) for about 12 min in the dark, washed in 0.1 M PB (pH 7.4) and subsequently incubated in 0.05% DAB (MilliporeSigma, Cat#D5637; RRID:AB_2336819) and 0.01% hydrogen peroxide prepared in 0.1 M PB for 3 min. Finally, the sections were osmicated (1% osmium tetroxide, Electron Microscopy Sciences, Cat#19150) in 0.1 M PB pH 7.4 for 20 min, washed in 0.1 M PB (pH 7.4), dehydrated in graded alcohols (50%–100%) to propylene oxide and plastic-embedded in Epon resin 812. 50–60 nm-ultrathin sections were cut with a diamond knife (Diatome USA), collected on nickel mesh grids, stained with 2.5% lead citrate, and examined with a JEOL JEM 1400 Plus electron microscope. Tissue samples were imaged using a digital camera (sCMOS). The double immunocytochemical method was repeated three times on the sections obtained from each of the three brains studied. Immunoperoxidase and immunogold-labeling were visualized on the hippocampal sections with a light microscope and portions of the CA3 stratum lucidum and the dentate hilus with good and consistent mGluR1b and mGluR5 receptors immunolabeling were identified and trimmed down for ultrathin sectioning. Three to four semi-thin sections (1 µm-thick) were then cut with a histo-diamond knife (Diatome USA) and stained with 1% toluidine blue. To further standardize the conditions, only the first 20 ultrathin sections (50–60 nm thick) were cut, collected onto the grids and photographed. The electron micrographs were taken at 8,000×with a Digital Morada Camera from Olympus (Hamburg, Germany). Sampling was always carefully and accurately carried out in the same way for the brains studied and it was blinded to experimenters during mGluRs quantification. Data analysis and statistics Electrophysiology and calcium imaging. Graphs and statistical analysis were performed using OriginPro (Origin Laboratory, Northampton, MA). The normality of data distribution was assessed with the Shapiro–Wilk test. Data with a normal Frontiers in Cellular Neuroscience 05 frontiersin.org fncel-17-1068472 March 30, 2023 Time: 15:35 # 6 Lutzu et al. 10.3389/fncel.2023.1068472 FIGURE 3 NMDAR and mGluR5 but not mGluR1 contributed to postsynaptic calcium rise evoked during presynaptic MF burst activity. (A) Calcium signals induced by presynaptic stimulation (PRE) before and after bath application of the NMDAR antagonist D-APV (25 µM). Representative traces (top) and summary plot of the averaged peak 1G/R before (normalized to baseline) and after drug application. (B–D) Same experimental design as in panel (A) but following application of the mGluR5 antagonist MPEP (4 µM) (B), the mGluR1 antagonist YM (1 µM) and in naïve conditions (i.e., calcium signals monitored during 15–30 min in the absence of drug applications) to evaluate stability. Data are presented as mean ±SEM. ***p<0.001, n.s. not significant. distribution were analyzed using a parametric test (One-Sample t Test for unpaired data and Paired t Test for paired data), whereas data that did not display a normal distribution were analyzed with a non-parametric test (Mann–Whitney). For multiple comparisons, we employed a One-Way ANOVA with a Bonferroni post hoc analysis for mean comparison. Statistical significance was set at P<0.05. Immunoelectron microscopy. The double immunocytochemical method was repeated three times on the sections obtained from each of the three brains that were analyzed. In particular, a total of 181 dendritic thorny excrescences (TEs) of pyramidal neurons contacted by MF terminals in CA3 stratum lucidum (area studied: 1406.1 µm2) and 153 mossy cell TEs with MF synapses in dentate hilus (area studied: 1406.1 µm2) were analyzed. Positive mGluR1b and/or mGluR5 TEs were identified by the presence of DAB immunodeposits and/or by the presence of at least one gold particle within 30 nm of the postsynaptic membrane. Collecting data of mGluR1b/mGluR5 positive TEs obtained for each antibody in CA3 and in the dentate hilus were pooled. The percentage of mGluR1b and/or mGluR5 positive TEs was analyzed and displayed using a statistical software package (GraphPad Prism 8, GraphPad Software Inc, San Diego, CA, USA; RRID:SCR_002798). Data are presented as mean ±SEM. The data were pooled since three analyzed samples did not differ in mGluRs labeling (Kolmogorov Smirnov test, P>0.19). Then, the ratio of the total mGluR5 versus total mGluR1b in TEs of CA3 and dentate hilus was compared. The normality test (Kolmogorov–Smirnov normality test) was applied before statistical tests and subsequently, data were analyzed using a parametric unpaired t Test. Drugs and chemicals All chemicals and drugs used were purchased from MilliporeSigma (St. Louis, MO, USA) except D-APV, NBQX, CGP-55845, DCG-IV, MNI-caged-L-glutamate and CPA which were obtained from Tocris Bioscience (Minneapolis, MN, USA), LY 303070 which was obtained from ABX advanced biochemical compounds (Radeberg, Germany), and Fluo5f and Alexa which were purchased from Invitrogen (Carlsbad, CA, USA). Results The magnitude of postsynaptic calcium rise during NMDAR-LTP and LTD induction differs To assess the postsynaptic calcium signals elicited by distinct patterns of activity that induce NMDAR-LTP and NMDARLTD at MF-CA3 synapses, we combined whole-cell patch clamp recordings and 2P calcium imaging in acute rat hippocampal slices. We patch-loaded CA3 pyramidal cells with a potassium-based internal solution containing the low-affinity calcium indicator Fluo5f (300 µM), and Alexa 594 (20 µM) which was used as a morphological indicator. We performed recordings in currentclamp configuration in the presence of picrotoxin (30 µM) to block GABAA-mediated transmission, and a low concentration of LY303070 (0.5–1 µM) to minimize AMPAR-mediated synaptic potentials arising from the recurrent associational-commissural network (Kwon and Castillo,2008b). Under these recording conditions, presynaptic MF stimulation (e.g., 5 stimuli, 50 Hz) evoked subthreshold EPSPs that were associated with calcium transients (CaTs) in the target postsynaptic CA3 spines thorny excrescences (TEs) (Figures 1A–C;1G/R PRE = 0.09 ±0.01) and, as expected, direct activation of CA3 pyramidal neurons (3 action potentials, 100 Hz) also induced CaTs in TEs (Figures 1A– C; POST = 0.12 ±0.01). As previously reported, activation of postsynaptic glutamate receptors, calcium release from the internal Frontiers in Cellular Neuroscience 06 frontiersin.org fncel-17-1068472 March 30, 2023 Time: 15:35 # 7 Lutzu et al. 10.3389/fncel.2023.1068472 stores, and activation of voltage-gated calcium channels likely accounts for these CaTs (Kapur et al.,1998,2001;Reid et al., 2001). We next analyzed CaTs elicited by single PRE-POST and POST-PRE pairings which are known to trigger burst timingdependent NMDAR-LTP and LTD, respectively (Hunt et al., 2013). Single pairings provide an estimation of the postsynaptic calcium changes occurring during the full induction protocol of plasticity (Koester and Sakmann,1998;Nevian and Sakmann, 2004;Mishra et al.,2016). We found that PRE-POST pairings elicited larger CaTs than POST-PRE pairings (Figures 1B, C,1G/R PRE-POST = 0.25 ±0.02; POST-PRE = 0.16 ±0.01; n= 13; PRE-POST vs POST-PRE: paired t Test p= 0.00026), indicating that the induction of NMDAR-LTP is likely associated with larger postsynaptic calcium rise than NMDAR-LTD. Extracellular MF stimulation could recruit extrinsic neuromodulatory fibers and trigger the release of modulators from the MF boutons, both of which could potentially affect postsynaptic CaTs. To directly address this issue, we employed 2P glutamate uncaging and tested whether glutamate alone could mimic the different CaTs following PRE-POST and POST-PRE pairing protocols with MF extracellular electrical stimulation. We found that 2P glutamate uncaging ∼1µm from TEs (Figure 1D) elicited remarkably similar PRE-POST and POST-PRE CaTs as observed with electrical stimulation (Figures 1E,F,1G/R uncaging PRE = 0.09 ±0.03; POST = 0.05 ±0.01; PRE-POST = 0.15 ±0.02; POST-PRE: 0.10 ±0.02; PRE-POST vs POST-PRE: paired t Test p= 0.0099). These results indicate that the different calcium signals elicited by PRE-POST and POST-PRE pairing protocols are due to the direct activation of postsynaptic glutamate receptors. To determine whether the difference in calcium signals observed with single pairings was preserved during the full induction protocol, which comprises 100 PRE-POST (NMDARLTP) or POST-PRE (NMDAR-LTD) pairings delivered at 2 Hz respectively (Hunt et al.,2013), we measured CaTs while delivering these protocols in the same cell (Figure 1G). An equal number of cells received a single LTP or LTD protocol first, and the second protocol was delivered 5 min after the first one (see Methods). We found that the NMDAR-LTP induction protocol elicited larger CaTs compared to the NMDAR-LTD induction protocol (Figures 1H,I). To quantify this difference, we calculated the integrated area under the curve in arbitrary units (a.u.) (Figures 1H, I, PRE-POST = 18,414.30 ±2,833.90 a.u.; POSTPRE = 12,229.05 ±1,964.84 a.u.; PRE-POST vs POST-PRE: n= 8, paired t Test, p= 0.00529). Altogether, our results indicate that NMDAR-LTP induction is associated with a larger calcium rise than NMDAR-LTD induction and that postsynaptic glutamate receptors most likely account for this difference. mGluR1 and mGluR5 contribute differently to postsynaptic calcium rise evoked by PRE-POST and POST-PRE patterns of activity At the MF-CA3 synapse, mGluR5 and mGluR1 are required for burst timing-induced NMDAR-LTP and LTD, respectively (Hunt et al.,2013). Because Gq-coupled receptors activate the phosphoinositide pathway that mediates calcium release from calcium stores (Niswender and Conn,2010), we hypothesized that co-activation of NMDAR and mGluR1 or mGluR5 determines the magnitude of the postsynaptic calcium that triggers MF-CA3 NMDAR-LTP and LTD. To test this hypothesis, we delivered the PRE-POST and POST-PRE stimulation pairings in the presence of mGluR1 or mGluR5 selective antagonists. As previously reported, mGluR1 and mGluR5 antagonism has no significant effect on basal MF-CA3 synaptic transmission (Hunt et al.,2013). In comparison to control conditions (Figure 2A, Data replotted from Figure 1C for comparison reasons), bath application of the non-competitive mGluR5 antagonist MPEP (4 µM) abolished the difference between PRE-POST and POST-PRE CaTs (Figure 2B, MPEP 1G/R PREPOST = 0.20 ±0.03; POST-PRE = 0.18 ±0.03; n= 12, paired t Test p= 0.347). Conversely, blockade of mGluR1 with the non-competitive antagonist YM298198 (1 µM) did not alter the difference between PRE-POST and POST-PRE observed in control conditions (Figure 2C, YM 1G/R PRE-POST = 0.25 ±0.04; POSTPRE = 0.16 ±0.04; n= 12, paired t Test: p= 0.0025). As positive control that YM was efficiently blocking mGluR1, in a separate set of experiments we found that the inward current mediated by the I-mGluR agonist DHPG (30 µM) in CA1 pyramidal neurons was significantly reduced by YM co-application, as previously reported (Mannaioni et al.,2001;Rae and Irving,2004) (Figure 2D, Control: −19.49 ±1.28 pA, n= 3; YM: −4.46 ±2.63 pA, n= 3; two sample t-test p= 0.0068). These results indicate that mGluR1 and mGluR5 contribute differently to postsynaptic calcium rise at MFCA3 synapses, consistent with their distinct role in NMDAR-LTP and LTD. Glutamate release during MF burst activity engages diverse postsynaptic calcium sources Previous studies at the MF-CA3 synapse reported that glutamate triggers postsynaptic calcium rise via activation of ionotropic receptors and/or via mGluR-mediated calcium release from intracellular stores (Jaffe and Brown,1997;Kapur et al., 2001;Reid et al.,2001). Of note, calcium signals in these studies were elicited by non-physiological tetanic stimulation (e.g., 100 Hz, 1 s) (Jaffe and Brown,1997), single stimulation in organotypic slice cultures (Reid et al.,2001), or were analyzed using a lowspatial resolution imaging approach (Kapur et al.,2001). We therefore tested the contribution of ionotropic glutamate receptors and mGluRs in generating postsynaptic calcium signals elicited by physiologically relevant MF burst stimulation (PRE only) and monitored using 2P calcium imaging in acute rat hippocampal slices. After obtaining baseline CaTs in response to a short burst of MF stimulation (5 pulses, 50 Hz), we blocked NMDARs with the competitive antagonist D-APV (25 µM). This manipulation nearly abolished postsynaptic CaTs (Figure 3A, PRE + DAPV = 0.10 ±0.02 1G/R normalized to control; n= 16, paired t Test: p= 0.000005), indicating that NMDARs are robustly engaged during MF burst activity and mediate most of the postsynaptic calcium rise despite being expressed at a lower level than AMPARs (Takumi et al.,1999). In separate experiments, the mGluR5 antagonist MPEP (4 µM) significantly reduced postsynaptic CaTs (Figure 3B, Frontiers in Cellular Neuroscience 07 frontiersin.org fncel-17-1068472 March 30, 2023 Time: 15:35 # 8 Lutzu et al. 10.3389/fncel.2023.1068472 FIGURE 4 Calcium depletion from internal stores significantly reduced the difference between PRE-POST and POST-PRE CaTs at MF-CA3 synapses. (A) Representative CaTs induced by PRE-POST and POST-PRE stimulation in the presence of the SERCA pump blocker cyclopiazonic acid (CPA) (top). Brain slices were incubated for 30 min in 30 µM CPA which was also continuously perfused. Summary plot showing the effect of CPA on peak 1G/R measured during PRE-POST and POST-PRE stimulation (bottom). (B) PRE-POST and POST-PRE CaT ratio under different experimental conditions: Ctrl (no drugs), 4 µM MPEP, 30 µM CPA and 1 µM YM. One Way ANOVA; F= 5.86458; p= 0.00194; DF = 3; Ctrl vs MPEP, p= 0.0036; Ctrl vs CPA, p= 0.0198; Ctrl vs YM, p= 0.136; Bonferroni post hoc analysis. (C) Non-linear summation of postsynaptic calcium signals under different experimental conditions. Non-linearity was measured as the ratio between the measured PRE-POST or POST-PRE CaTs and the arithmetical sum of the PRE and POST components alone. Analysis was performed on data shown in Figures 2B–D,4A. All data points were compared against a non-linearity factor of 1 using One Sample t Test. Data are presented as mean ±SEM. **p<0.01, *p<0.05, n.s. not significant. PRE + MPEP = 0.49 ±0.04 1G/R normalized to control, n= 11, paired t Test: p= 0.000041), suggesting that MF burst activity increases postsynaptic calcium via mGluR5 and NMDAR coactivation. In contrast, mGluR1 antagonism with YM (1 µM) had no significant effect on CaTs (Figure 3C, PRE + YM = 0.84 ±0.08 1G/R normalized to control, n= 12. paired t Test: p= 0.139). Likewise, no significant changes were observed in naïve slices during 15–30 min, i.e., after no drug application (Figure 3D, PRE – 15–30 min = 1.02 ±0.13 normalized 1G/R, n= 11, Wilcoxon signed ranks test: p= 0.83). Overall, our results strongly suggest that mGluR5 and mGluR1 engage different intracellular cascades implicated in MF-CA3 postsynaptic calcium rise. Intracellular calcium stores determine the calcium rise magnitude elicited by NMDAR-LTP and LTD induction The postsynaptic calcium rise mediated by mGluRs is likely due to their positive coupling to calcium release from internal stores, such as the endoplasmic reticulum (ER) (Kapur et al., 2001;Reid et al.,2001). Therefore, we predicted that depletion of calcium from the ER should reduce the difference between single PRE-POST and POST-PRE pairings. Indeed, this difference was significantly reduced in hippocampal slices incubated for at least 30 minutes with the SERCA pump blocker cyclopiazonic acid (CPA, 30 µM) which depletes the calcium content from the ER (Figure 4A,1G/R PRE-POST = 0.26 ±0.04; 1G/R POSTPRE = 0.22 ±0.03; PRE-POST vs POST-PRE: n= 9, Paired t Test p= 0.01116; see Figure 2A). To better quantify the effect of CPA, we calculated the PRE-POST/POST-PRE ratio of CaTs, which allows the comparison across different recording conditions. This analysis revealed that mGluR5 antagonism and/or CPAmediated depletion of calcium stores, but not mGluR1 antagonism, abolished the difference between PRE-POST and POST-PRE CaTs (Figure 4B, PRE-POST/POST-PRE; Ctrl = 1.62 ±0.13, n= 13; MPEP = 1.15 ±0.07 n= 12; CPA = 1.20 ±0.06, n= 9; YM = 1.45 ±0.07, n= 12; One Way ANOVA, F= 5.865; p= 0.00194; DF = 3.; Ctrl vs MPEP p= 0.0036; Ctrl vs CPA p= 0.0198; Ctrl vs YM p= 0.136; Bonferroni post hoc test). These results indicate that the PRE-POST, but not the POST-PRE protocol engages mGluR5 signaling and calcium release from intracellular stores, two key determinants of the distinct CaTs between NMDAR-LTP and LTD. Pairing of presynaptic and postsynaptic action potentials with short delays can elicit non-linear summation of postsynaptic calcium signals (Yuste and Denk,1995;Koester and Sakmann, 1998), which is thought to be important for bidirectional plasticity of AMPAR-mediated transmission (Nevian and Sakmann,2004, 2006). To test for potential non-linear summation of postsynaptic calcium signals generated by MF-CA3 PRE-POST and POST-PRE pairing under different conditions, we performed a non-linearity analysis by calculating a non-linearity factor (NLF), which is the ratio between the measured PRE + POST summation (i.e., PREPOST CaTs peak) and the predicted PRE + POST summation (i.e., the arithmetical sum between PRE and POST peak CaTs) (Nevian and Sakmann,2004). We found that under control conditions, the PRE-POST pairing is supralinear (i.e., NLF >1), while the POST-PRE coupling is significantly sublinear (i.e., NLF <1) (Figure 4C). Consistent with a critical role of mGluR5 and their downstream targets calcium stores in the generation of different CaTs levels between PRE-POST and POST-PRE, both MPEP and CPA abolished the supralinear summation of PRE and POST CaTs. In contrast, the blockade of mGluR1 with YM, did not Frontiers in Cellular Neuroscience 08 frontiersin.org fncel-17-1068472 March 30, 2023 Time: 15:35 # 9 Lutzu et al. 10.3389/fncel.2023.1068472 affect the non-linearity factor of the PRE-POST and POST-PRE pairings, (Figure 4C, NLF Ctrl PRE-POST = 1.22 ±0.07, n= 13, p= 0.01; Ctrl POST-PRE = 0.78 ±0.05, n= 13, p= 0.003; MPEP PRE-POST = 0.97 ±0.06, n= 12, p= 0.72; MPEP POSTPRE = 0.87 ±0.05, n= 12, p= 0.054; YM PRE-POST = 1.20 ±0.05, n= 12, p= 0.002; YM POST-PRE = 0.84 ±0.03, n= 12, p= 0.001; CPA PRE-POST = 0.90 ±0.07, n= 9, p= 0.16; CPA POSTPRE = 0.75 ±0.05, n= 9, p= 0.001; One Sample t Test, all comparisons were against the value NLF = 1; Ctrl PRE-POST vs POST-PRE, p= 0.00001; MPEP PRE-POST vs POST-PRE, p= 0.91; CPA PRE-POST vs POST-PRE, p= 0.846; YM PRE-POST vs POST-PRE, p= 0.0011; One-Way ANOVA, F= 9.19144; DF = 7; Bonferroni post hoc test). Taken together these results suggest that mGluR5, but not mGluR1, activation and calcium release from the internal stores are critical for the larger calcium rise associated with the induction of NMDAR-LTP. NMDAR plasticity at the MF-to-Hilar mossy cell synapse MFs also make synapses with hilar mossy cells (MCs), and these synapses share structural and functional properties with MFCA3 synapses, including the presence of giant MF boutons and postsynaptic TEs, a high degree of synaptic facilitation, regulation of glutamate release by mGluR2/3 agonists and the expression of presynaptic LTP (Lysetskiy et al.,2005;Scharfman,2016). However, whether MF-MC synapses can undergo NMDAR plasticity was never tested. We therefore recorded MCs and delivered PRE-POST and POST-PRE parings like for MF-CA3 synapses. As previously reported, MCs in rat hippocampal slices were identified by the high frequency of spontaneous synaptic activity, regular action potential firing, and minimal-to-no afterhyperpolarization (Larimer and Strowbridge,2008). In addition, MCs were patch-loaded with Alexa 594 and their identity was confirmed post hoc by the visualization of TEs (Figure 5A). We found that the PRE-POST pairing protocol induced robust NMDAR-LTP at MF-MC (Figure 5B, MC PREPOST induction = 174 ±14% of baseline; n= 6; p= 0.00075; paired t Test). In contrast, the POST-PRE protocol did not trigger NMDAR-LTD, but a small, yet significant, LTP (Figure 5B, MC POST-PRE induction = 121 ±4% of baseline; n= 7; p= 0.0021; paired t Test). Importantly, the PRE-POST protocol did not induce LTP of the AMPAR-mediated component of MF-MC synaptic transmission (Figure 5C, AMPAR EPSC = 111 ±9% of baseline, n= 6; Ctrl vs AMPAR p= 0.00089, One Way ANOVA). The selective strengthening of the NMDARbut not AMPAR-mediated component supports a postsynaptic mechanism of NMDAR-LTP expression at MF-MC synapses. To determine whether MF-MC NMDAR-LTP required postsynaptic calcium rise, we first loaded MCs with the calcium chelator BAPTA (20 mM). This manipulation significantly reduced the magnitude of NMDAR-LTP (Figure 5C, Control MC NMDAR LTP = 180 ±7% of baseline, n= 9; NMDAR LTP + BAPTA = 120 ±15% of baseline, n= 6; Ctrl vs BAPTA p= 0.00442, One Way ANOVA; F= 10.41736; p= 0.0000161; DF = 4; Bonferroni post hoc). Given the critical role of I-mGluRs for the induction of NMDAR-LTP at most central synapses (Hunt and Castillo,2012), including the MF-CA3 synapse FIGURE 5 NMDAR plasticity at the MF-to-Hilar Mossy Cells synapse. (A) Representative 2P reconstruction of a MC patch-loaded with biocytin (left). MCs displayed regular action potential firing induced by current injection and minimal to no after hyperpolarization (right). (B) Both PRE-POST and POST-PRE pairing protocols induced NMDAR LTP but not LTD at MF-to-Hilar Mossy Cells (MF-MC) synapses. (C) MF-MC NMDAR LTP induced by PRE-POST pairing under different experimental conditions: control, 20 mM intracellular BAPTA, 1 µMYMand4µM MPEP, and 30 µM CPA. The PRE-POST pairing protocol had no effect on AMPAR-mediated synaptic transmission. One Way ANOVA; F= 9.19144; p= 0.0000161; DF = 4.; Ctrl vs BAPTA p= 0.00442; Ctrl vs YM p>0.05; Ctrl vs MPEP p= 0.0243; Ctrl vs CPA p= 0.04. Bonferroni post hoc analysis. (D) Representative MC TE depicting the line scan (yellow line) where 2P calcium imaging was performed (top left), PRE-POST vs POST-PRE MF-MC calcium signals (bottom left), and summary plot (right). Data are presented as mean ±SEM. **p<0.01, *p<0.05, n.s. not significant. Frontiers in Cellular Neuroscience 09 frontiersin.org