# Astrocytic EphB3 receptors regulate d-serine-gated synaptic plasticity and memory

Valentin Clément Langlais, Sarah Mountadem, Ines Benazzouz, Aurélie Amadio, Marco Matos, Aurélie Jourdes, Astrid Cannich, Francisca Julio-Kalajzic, Ilaria Belluomo, Isabelle Matias, et al.

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## To cite this version:

Valentin Clément Langlais, Sarah Mountadem, Ines Benazzouz, Aurélie Amadio, Marco Matos, et al. Astrocytic EphB3 receptors regulate d-serine-gated synaptic plasticity and memory. Progress in Neurobiology, 2025, 248, pp.102747. 10.1016/j.pneurobio.2025.102747. hal-05405082

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# Astrocytic EphB3 receptors regulate D-serine-gated synaptic plasticity and memory

Valentin Clément Langlaisª, Sarah Mountademª, Ines Benazzouzª, Aurélie Amadioª, Marco Matosª, Aurélie Jourdesª, Astrid Cannichª, Francisca Julio-Kalajzicª,ᵇ,ᶜ, Ilaria Belluomoª, Isabelle Matiasª, Marlène Maitreª, Thierry Lesté-Lasserreª, Sébastien Maraisᵇ, Elena Avignoneª, Giovanni Marsicanoª, Luigi Bellocchioª, Stéphane Henri Richard Olietª, Aude Panatierª,ᶜ,ᶜ,ª

## ARTICLE INFO

**Keywords:**
Astrocyte
D-serine
Ephrin
LTP
Memory
NMDA receptor

## ABSTRACT

The activation of classical NMDA receptors (NMDARs) requires the binding of a co-agonist in addition to glutamate. Whereas astrocytic-derived D-serine was shown to play such a role at CA3-CA1 hippocampal synapses, the exact mechanism by which neurons interact with neighboring astrocytes to regulate synaptic D-serine availability remains to be fully elucidated. Considering the close anatomical apposition of astrocytic and neuronal elements at synapses, the aforementioned process is likely to involve cells adhesion molecules. One very likely candidate could be the astrocytic EphB3 receptor and its neuronal partner, ephrinB3. Here, we first showed in acute hippocampal slices from adult mice that stimulation of EphB3 receptors with exogenous ephrinB3 increased D-serine availability at CA3-CA1 synapses, resulting in an increased NMDAR activity. Conversely, inhibiting endogenous EphB3 receptors caused an impairment of both synaptic NMDAR activity and NMDAR-dependent long-term synaptic potentiation (LTP), effects that could be rescued by exogenous D-serine. Most interestingly, knocking down EphB3 receptors specifically in astrocytes yielded a similar impairment in hippocampal plasticity and, most importantly, caused a deficit in novel object recognition memory. Altogether, our data thus indicate that EphB3 receptors in hippocampal astrocytes play a key role in regulating synaptic NMDAR function, activity-dependent plasticity and memory.

## 1. Introduction

Long-term synaptic plasticity is considered as the cellular substrate of learning and memory (Lynch, 2004). This includes long-term potentiation (LTP), which corresponds to a persistent increase of synaptic strength (Bear and Malenka, 1994; Bliss and Collingridge, 2013; Bliss and Gardner-Medwin, 1973; Bliss and Lomo, 1973; Nicoll, 2017). The most common form of synaptic plasticity in the brain depends on N-methyl-D-aspartate receptors (NMDARs) (Citri and Malenka, 2007; Collingridge et al., 1983) whose activation requires the binding of both glutamate and a co-agonist (Johnson and Ascher, 1987; Kleckner and Dingledine, 1988) that was identified to be D-serine at hippocampal CA3-CA1 synapses (Henneberger et al., 2010; Mothet et al., 2000;

Papouin et al., 2012). D-serine is provided locally by the astrocytic environment (Fossat et al., 2012; Panatier et al., 2006; Papouin et al., 2012; Sherwood et al., 2017, 2021; Yang et al., 2003) in a Ca²⁺ dependent manner (Henneberger et al., 2010; Mothet et al., 2005; Sherwood et al., 2017; Shigetomi et al., 2013). The detailed mechanism responsible for the neuron-to-astrocyte crosstalk leading to synaptic D-serine availability is not completely understood. While astrocytic CB1 receptors have been shown to be involved in this intercellular communication (Fernández-Moncada et al., 2024; Robin et al., 2018), another important factor is the intimate anatomical relationship between the thin astrocytic processes and the synaptic neuronal elements (Panatier et al., 2006). Interestingly, crosstalk signaling between astrocytes and neurons can occur through the interaction of cell adhesion molecules

<footnote>ª Univ. Bordeaux, INSERM, Neurocentre Magendie, U1215, Bordeaux F-33000, France</footnote>

<footnote>ᵇ Univ. Bordeaux, CNRS, INSERM, BIC, US4, UAR 3420, Bordeaux F-33000, France</footnote>

<footnote>ᶜ Lead contact, France</footnote>

<footnote>* Corresponding author at: Univ. Bordeaux, INSERM, Neurocentre Magendie, U1215, Bordeaux F-33000, France.
E-mail address: aude.panatier@inserm.fr (A. Panatier).</footnote>
(CAMs), which depends on the close proximity of the astrocytic element with its synaptic partners (Dalva et al., 2007; Spacek and Harris, 1998; Thalhammer and Cingolani, 2014; Togashi et al., 2009).

Among CAMs, increasing evidence suggests the participation of the family erythropoietin-producing hepatocellular carcinoma (Eph) receptors (Hirai et al., 1987) and their ligands, ephrins, in the bidirectional communication between astrocytes and neurons (Klein, 2009; Murai and Pasquale, 2011). Previous works have suggested that astrocytic EphB3 receptors could play a role in the regulation of astrocytic D-serine release (Zhuang et al., 2011, 2010). Indeed, stimulation of EphB3 receptors increased D-serine concentration in pure astrocytic cultures as well as in CA1 hippocampal tissue (Zhuang et al., 2010). Strikingly, knocking out EphB3 receptors or ephrinB3 ligands in all cell types led to impaired LTP (Rodenas-Ruano et al., 2006; Zhuang et al., 2010). These studies, however, did not address the impact of EphB3 receptor or ephrinB3 ligand on NMDARs functions. Thus, a very attractive hypothesis could be that the interaction between astrocytic EphB3 receptor and neuronal ephrinB3 ligand regulates synaptic D-serine availability, thereby modulating synaptic NMDAR activity, NMDAR-dependent LTP and ultimately governing memory.

In the present study we tested this hypothesis by showing that the EphB3 receptors activation leads to increased synaptic D-serine availability and NMDARs activity at CA3-CA1 synapses. Furthermore, inhibiting endogenous EphB3-ephrinB3 interaction had the opposite effect, decreasing D-serine availability and impairing NMDAR-responses and LTP. Finally, we showed that, by controlling D-serine availability, astrocytic EphB3 receptors control LTP induction as well as novel object recognition memory. Taken together, our findings reveal that EphB3-ephrinB3 interactions at the tripartite synapse regulate excitatory transmission, long-term synaptic plasticity and novel object recognition consolidation through the control of NMDAR co-agonist-binding site occupancy.

## 2. Materials and methods

### 2.1. Animals

All experiments were performed in C57BL-6J male mice (Janvier, Le Genest Saint Isle, France). Animals were housed in standard cages with a 12-h light/dark cycle and free access to food and water. All the experiments involving animals were conducted in strict compliance with the European directive (2010/63/EU) and after approval of ethical committee (approval number 16,161 and 20,053).

### 2.2. Acute slices preparation

Adult C57BL-6J male mice (8–14 weeks old) were anesthetized with 5% isoflurane inhalation for 2 min (2 L/min) and decapitated. The brain was quickly removed and transferred in ice-cold artificial cerebrospinal fluid (ACSF) saturated with 95% O₂ and 5% CO₂. ACSF composition was (in mM): 125 NaCl, 2.5 KCl, 1 Na₂HPO₄, 26 NaHCO₃, 1.2 MgCl₂, 0.6 CaCl₂ and 11 glucose (pH 7.3; 305 mosmol/kg). Coronal hippocampal slices (300–350 µm) were prepared using a vibratome (Microm HM 650 V; Leica VT1200S) and hemisected. Next, they were incubated at 33 °C during 30 min in ACSF containing 2 mM MgCl₂ and 1 mM CaCl₂. To recover, slices were then allowed to rest at least 1 h at room temperature (RT).

### 2.3. Electrophysiology

Experiments were performed at RT. Slices were placed into a recording chamber perfused with ACSF (2.8 ml/min) containing this time 1.3 mM MgCl₂ and 2.5 mM CaCl₂. Stimulation of the Schaffer collaterals (0.05 Hz, 100 µs duration) via a concentric bipolar tungsten electrode was used to induce synaptic responses. This electrode was located 200 µm away from the recording electrode placed in the *stratum radiatum* of CA1 area. Responses were recorded with a Multiclamp 700 A amplifier (Axon Instruments, Inc.). Signals were filtered (2 kHz; Multiclamp 700 A) and digitized (10 kHz; DigiData 1322 A, Axon Instruments, Inc.). Data were stored for offline analysis using pClamp9 software (Axon Instruments, Inc.).

**Extracellular fields recordings.** Field excitatory postsynaptic potentials (fEPSPs) were recorded using glass pipettes (2–4 MΩ) filled with ACSF. Electrophysiological recordings were carried out in the presence of picrotoxin (100 µM). Therefore, to suppress epileptiform activity, a cut between CA3 and CA1 was made. NMDAR activity recordings were performed in the same ACSF, but this time in low Mg²⁺ ACSF (0.2 mM) with 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline-2,3-dione (NBQX, 10 µM) to inhibit AMPA and kainate receptors. ACSF composition was (in mM): 125 NaCl, 2.5 KCl, 1 Na₂HPO₄, 26 NaHCO₃, 0.2 MgCl₂, 0.6 CaCl₂ and 11 glucose (pH 7.3; 303 mosmol/kg). Slopes were measured as a linear fit of the rising phase corresponding to 20 % and 60 % of the peak amplitude for fEPSPs and in between 20 % and 80 % for NMDAR-fEPSPs slopes. Paired-pulse ratio is the result of the slope of the second fEPSP (150 ms interval) divided by the slope of the first fEPSP. For LTP experiments, the intensity of the stimulation was set to 30–40 % of that triggering population spikes. Following a stable baseline (at least 10 min), LTP was induced by the use of a high-frequency stimulation (HFS) protocol. It consisted of three trains of stimuli (100 Hz, 1 sec duration) each separated by 20 sec. Representative fEPSPs and NMDAR-fEPSPs traces are the average of successive sweeps that correspond to 10 min and 20–30 min of stable recording, respectively. For clarity, stimulation artifacts were deleted from these representative traces. For post-tetanic potentiation (PTP) fEPSP slopes were measured just after HFS (0–1 min) relative to baseline (–10–0 min) and for LTP fEPSP slopes were measured after HFS (50–60 min) relative to baseline (–10–0 min).

**Astrocytic patch-clamp recordings.** Astrocytes in the *stratum radiatum* were identified using infrared DIC microscopy (Olympus BX50WI) based on soma size (≤ 10 µm), shape, and biophysical properties. First, astrocytes were characterized by performing a ramp voltage command from –120 to +100 mV in voltage-clamp mode. The current-voltage relationship had to be linear. Second, steps of voltage induced by square pulses of currents (800 pA steps, 600 ms duration) in current-clamp configuration didn't induce action potential. Finally, resting membrane potential of the recording cell (recorded in current-clamp configuration) was never higher than –70 mV during all the experiment. Patch-clamp recording pipettes (5–6 MΩ) were filled with (in mM): 130 KMeSO₃, 10 HEPES, 10 Sodium phosphocreatine, 4 Na₂-ATP, 0.4 Na-GTP and 4 MgCl₂ (pH 7.2; 300 mosmol/kg). To constrain free astrocytic Ca²⁺, we used a Ca²⁺-clamp internal solution, to clamp the Ca²⁺ concentration at 50–80 nM, by adding (in mM): 0.2 BAPTA, 0.45 EGTA and 0.14 Ca²⁺ to the intracellular solution as previously done (Henneberger et al., 2010). At the end of each experiment, AP5 (50 µM) was applied to isolate the remaining non-NMDAR component of the response; which was then subtracted from the responses to obtain pure astrocytic NMDAR-fEPSPs (a-NMDAR-fEPSPs). Average a-NMDAR-fEPSPs traces correspond to 10 min of stable recording. For clarity the stimulation artifact was deleted.

### 2.4. Laser-induced fluorescence capillary electrophoresis

5 hippocampi from adult C57BL-6J mice (8–14 weeks old) were
isolated from 350 µm slices and incubated in 350 µl oxygenated ACSF with either clustered IgG1-Fc (0.27 µg/ml) or ephrinB3-Fc (0.5 µg/ml) during 1 h at room temperature. Extracellular medium was quickly extracted, frozen using liquid nitrogen and stored at -80 °C. Extracellular levels of D-serine and L-glutamate were then determined. Briefly, pooled slices were deproteinized by addition of cold trichloroacetic acid (TCA) to a 4% final concentration. The suspension was centrifuged at 10,000 g for 10 min, the TCA was extracted from the supernatant with water-saturated diethyl ether and stored at -80 °C. Samples were analyzed with a commercial laser-induced fluorescence capillary electrophoresis (CE-LIF) (CE: Beckman Coulter (Brea, California, US), P/ACE MDQ; LIF: Picometrics (Labège, France), LIF-UV-02, 410 nm 20 mW) as following: samples were processed for micellar CE-LIF and were fluorescently derivatized at RT for 60 min with napthalene-2,3-dicarboxaldehyde (NDA) before being analyzed by CE using a β-cyclodextrin (β-CD) based chiral separation buffer. All electropherograms data were collected and analyzed using Karat 32 software v8.0 (Beckman Coulter). The extracellular amounts of D-serine and L-glutamate were normalized to the protein content determined from pooled hippocampal slices by the Lowry method using the BCA protein Pierce (ThermoScientific, CA) assay with bovine serum albumin (BSA) as standards. The quantity of D-serine and L-glutamate in the samples was determined from a standardized curve while peak identification was made by spiking the fraction with the amino acid.

## 2.5. Design and ephb3 shRNA

We used the Invitrogen and pSM2 design (http://cancan.cshl.edu/RNAi_central/) algorithms to select shRNAs directed against the mouse ephb3 gene coding or 3'UTR regions. To express the shRNAs, we used the pPRIME system (Stegmeier et al., 2005). This generates micro-RNA-30 (mir30)-derived shRNAs and co-expression of a marker protein, such as DsRed, from the same transcript. The shRNA hairpin sequences were placed into the mir30 site of pPRIME-cmv-DsRed-FF3 (Addgene Plasmid 11,664). The DNA sequences of the hairpins (21 nucleotides) were:

sh-Ephb3: 5'- TGCTGTTGACAGTGAGCGCCCACTCAAGCTCTACT GCAATTAGTGAAGCCACAGATGTAATTGCAGTA-GAGCTTGAGTGGTTGCCTACTGCCTCGGA-3'

sh-control: 5'- TGCTGTTGACAGTGAGCGAGCCCACTACGTGAACCA TCACTAGTGAAGCCACA-GATGTAGTGATGGTTCACGTAGTGGGCCTGCCTACTGCCTCGGA-3'.

The hairpin oligonucleotides were PCR amplified by VENT polymerase (NEB, UK) using the pPRIME forward and reverse oligonucleotides with added XhoI and EcoRI sites underlined (forward: 5'-GATGGCTG-CTCGAG-AAGGTATAT-TGCTGTTGACAGTGAGCG-3'; reve rse, 5'-GTCTAGAG-GAATTC-CGAGGCAGTAGGCA-3'), digested with XhoI and EcoRI, and inserted into the mir30 site of EcoRI/XhoI-digested pPRIME (Zhang et al., 2015). To test knockdown efficiency of the shephb3 hairpins, STQ7 striatal cells were nucleofected with the pPRIME-DsRED-mir30 plasmids as previously described (Bellocchio et al., 2016). 48 h afterwards, RNA was extracted and knockdown efficiency was analyzed by measuring ephb3 gene expression as described later.

## 2.6. Generation of AAV-GFAP-DsRED-mir30-shephb3

To generate AAVs for the selective astrocytic expression of miRNAs, the DsRed-mir30-shephb3, and DsRED-mir30-sh-control inserts in

pPRIME were released by Nhel and HindIII digestion, and cloned into the polylinker of the AAV-genome plasmid pAAV.GFAP.eGFP.WPRE.hGH (Addgene Plasmid 50,473), in order to express DsRED-mir30-sh under the control of short gfaABC1D promoter (Lee et al., 2008). A sh-control was created more recently and we confirmed that the expression of a shRNA scramble had no impact neither on LTP, nor on novel object recognition memory memory (Fig. S4). These constructs were packed into AAV capsids serotype 8 by PEI transfection of 293 T cells and subsequent iodixalol gradient purification as previously described (Bellocchio et al., 2016; Hebert-Chatelain et al., 2016; Robin et al., 2018). Viral titers were 2.99 × 10¹¹gc/ml for AAV-GFAP- DsRED-mir30-shEphb3 and 2.71 × 10¹¹gc/ml for AAV-GFAP-DsRED-mir30-sh-control and 1.61x10¹¹gc/ml for pAAV.GFAP.eGFP as measured by digital PCR on viral DNA.

Specificity of astrocytic viral expression was confirmed by using q-PCR and immunohistochemistry as described after (Fig. S3).

**Laser Capture Microdissection (LCM) Analysis.** Brains were quickly extracted, flash-frozen in a vial of isopentane that was immerged in dry ice and subsequently stored at -80 °C until sectioning. Coronal Section (6 µm thickness) were mounted on polyethyl-ene-naphthalate membrane 1 mm glass slides (P.A.L.M. Microlaser Technologies AG, Bernried, Germany) that have been pretreated to inactivate RNase. Subsequently, sections were dehydrated in a series of pre-cooled ethanol baths (40 s in 95 % and twice 40 s in 100 %) and air-dried. Laser microdissection of cell was performed using a PALM MicroBeam microdissection system version 4.6 equipped with the P.A.L.M. RoboSoftware (P.A.L.M. Microlaser Technologies AG, Bernried, Germany). Laser power and duration were adjusted to optimize capture efficiency. Microdissection was performed at 5X to identify the CA1 Stratum radiatum and at 63X magnification to captured cells. A total between 800/1000 astrocytes (positives GFP or DsRed cells) per mice were captured. Cells were collected in adhesives caps and re-suspended PK buffer (PK buffer from RNeasy FFPE Kit, Qiagen, Chatsworth, USA), and stored at -80 °C until extraction was done. Total RNA was extracted from microdissected tissues using the RNeasy® FFPE Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The integrity of the RNA was checked by capillary electrophoresis using the RNA 6000 Pico Labchip kit and the Bioanalyser 2100 (Agilent Technologies, Massy, France), and quantity was estimated by spectrophotometry using a Denovix DS-11 (Denovix, Wilmington, USA).

**Quantitative Real-Time PCR (q-PCR).** RNA was processed and analyzed according to an adaptation of published methods (Bustin et al., 2009). Briefly, cDNA was synthesized from total RNA by using qSript™ cDNA SuperMix (Quanta Biosciences). qPCR was performed with a LightCycler® 480 Real-Time PCR System (Roche, Meylan, France). qPCR reactions were done for each sample by using LightCycler 480 SYBR Green I Master (Roche) in a final volume of 10 µl. The qPCR data were exported and analyzed in an informatics tool (Gene Expression Analysis Software Environment) developed at the NeuroCentre Magendie. The Genorm method was used to determine the reference gene (Bustin et al., 2009). Relative expression analysis was normalized against two reference genes. In particular, glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and peptidylprolyl isomerase A (Ppia) were used as reference genes. The relative level of expression was calculated with the comparative (2⁻ΔΔCT) method (Livak and Schmittgen, 2001). Primer sequences are reported in the table below.
<table><thead><tr><td>Gene</td><td>GenBank ID</td><td>Forward Sequence (5'-3')</td><td>Reverse Sequence (5'-3')</td></tr></thead><tbody><tr><td>Gapdh</td><td>NM_008084</td><td>TCAAGAAGGTGGTGAAGCAG</td><td>TGGGAGTTGCTGTTGAAGTC</td></tr><tr><td>Ppia</td><td>NM_008907</td><td>CAAATGCTGGACCAAACACAA</td><td>GCCATCCAGCCATTCAGTCT</td></tr><tr><td>Ephb2</td><td>NM_001290753</td><td>GAACGACCCTACTGGGACAT</td><td>GGGCGGAGGTAGTCTGTAGT</td></tr><tr><td>Ephb3</td><td>NM_010143</td><td>CGTCCAGTCATGATCCTCACT</td><td>GTACTTCATGCCAGCAGCAAT</td></tr><tr><td>Gad1</td><td>NM_008077</td><td>CCTTCGAGGGGTTCCAGATA</td><td>GAGGCTGGTAGCCCACCAT</td></tr><tr><td>Gfap</td><td>NM_010277</td><td>AACCGCATCACCATTCCCTGTA</td><td>CCACAGTCTTTACCACGATGTT</td></tr><tr><td>Rbfox3</td><td>NM_001039167</td><td>ACACACCCGCACAGACTCAT</td><td>GGGTCGGAAGGGTGGAGTT</td></tr><tr><td>S100 β</td><td>NM_009115</td><td>AAGTCAGAACTGAAGGAGCTTA</td><td>ACACTCCCCATCCCC ATCTT</td></tr></tbody></table>

## 2.7. Immunohistochemistry

To verify the correct pattern of DsRED expression and localization, mice were transcardially perfused with 4% paraformaldehyde (Sigma Aldrich, France) in PBS and brains were collected and sliced with a vibratome. Forty micrometer hippocampal sections incubated with primary antibody directed against S100β (1:1000, Rabbit polyclonal, Sigma Aldrich # HPA015768, France) and NeuN (1:500, Mouse monoclonal, Millipore # MAB377, France) overnight at 4 °C. Secondary antibodies incubation was performed in order to detect S100β with Alexa 488 (1:500, Thermo Scientific, France) and NeuN with Alexa 647 (1:500, Thermo Scientific, France) for 1 h at room temperature. Single plane confocal images were acquired with an SPE confocal microscope (Leica, France) and minimally processed with ImageJ software. Automatic quantification of DsRED, S100β and NeuN expressing cells was performed with ImageJ software as previously described (Bolte and Cordelieres, 2006; Robin et al., 2018). Briefly, after threshold subtraction and crosstalk correction, the number of cells co-expressing DsRED/S100β or DsRED/NeuN was automatically obtained by the “particle analysis” tool of the same software. DsRED/S100β co-expressing cells were expressed in percentage of CRE positive cells as well as percentage of total S100β cells. On the other hand, DsRED/NeuN co-expressing cells were reported as percentage of DsRED positive cells as well as percentage of total NeuN cells (Fig. S3C and S3D).

## 2.8. Stereotaxic injection

In vivo stereotaxic injections were performed in the CA1 area of the hippocampus of 7 weeks old male C56BL-6J mice in order to perform either electrophysiological experiments on acute slices or behavior 5 weeks later. During the entire procedure, each mouse was anaesthetized with isoflurane (induction at 4% + air 2 L/min, maintenance at 1.5% + air 0.3 L/min) and placed on a heating pad at 38 °C to maintain a stable body temperature. A subcutaneous injection of Meloxicam (1 mg/kg) was performed to reduce inflammation and pain caused by the surgery. Local anesthesia was performed using subcutaneous injection of lidocaine 0.5% before incision of the skull skin. Then a skin incision was performed and a hole was drilled in the skull to inject in each hemisphere 1 µL of viral solution with a Hamilton syringe at 0.2 µL/min. Virus were injected in the CA1 region of the hippocampus (coordinates from Bregma: anteroposterior: -1.67; lateral: +/- 1.25; ventral: -1.5 mm from the dura). The syringe was left in place for 5 min following each injection to minimize back flow of viral solution. After the injection, the skin was sutured using an ETHILON non-absorbable 3.0 and the mouse was placed in a post-operative recovery chamber maintained at 30 °C. Mice were then monitored until they wake up then put back in a collective box enriched with lab nest.

## 2.9. Novel object-recognition memory task

As before (Robin et al., 2018) we used the novel object recognition

memory task in a L-maze (Busquets-Garcia et al., 2013, 2011; Puighermanal et al., 2013, 2009). The L-shaped maze reduces the time of the assay while improving reliability of the measurements by increasing the exploratory behavior and avoiding possible anxiety-related bias of the open field environment (Da Cruz et al., 2020). The L-shaped maze is made of dark gray polyvinyl chloride shaped by two identical perpendicular arms (35 cm and 30 cm long respectively for external and internal L walls, 4.5 cm wide and 15 cm high walls) placed on a white background. The maze was overhung by a video camera allowing the detection and scoring online of animal's behavior as previously described (Hebert-Chatelain et al., 2016) (Robin et al., 2018). The task consisted of 3 sequential trials of 9 min each, one per day. During the habituation session (day 1, trial 1), mice were placed in the center of the maze and allowed to freely explore the arms in the absence of any objects. The acquisition session (day 2, trial 2) consisted in placing the mice again in the center of the maze in the presence of two identical objects positioned at the extremities of each arm and left to freely explore the maze and the objects. Right after the acquisition session, mice were injected intraperitoneally with D-serine (50 mg/kg in saline solution) or saline as vehicle control (Robin et al., 2018). Indeed, as reported before, D-serine is efficient to pass the BBB (Otte et al., 2013). In addition, we showed previously that the i.p. D-serine injection rescuing effect in GFAP-CB1-KO mice was also reproduced by intra-hippocampal injection of D-serine (Robin et al., 2018). The memory test occurred 24 h later (day 3, trial 3): one of the familiar objects was replaced by a novel object different in its shape, color, and texture and mice were left to explore both objects (Hebert-Chatelain et al., 2016; Robin et al., 2018). The apparatus as well as objects were cleaned with EtOH (20%) before experimental use and between each animal testing.

Memory performance was assessed by the discrimination index (DI) (Da Cruz et al., 2020). The DI was calculated as the difference between the time spent exploring the novel (TN) and the familiar object (TF) divided by the total exploration time (TN+TF): DI = [TN-TF]/[TN+TF]. In case of similar time exploring both novel and familiar object the DI will be proximal to 0, whereas a DI proximal to 0.5 indicate that mice spent around the triple of time exploring the novel versus the familiar object (Da Cruz et al., Bio Protoc, 2020). Memory was also evaluated by directly comparing the exploration time of novel and familiar objects, respectively. Object exploration was defined as the orientation of the nose to the object at a distance of less than 2 cm. Experienced investigators evaluating the exploration were blind of treatment and/or genotype of the animals.

## 2.10. Drugs

AP5 (50 µM), BAPTA (200 µM), DL-TBOA (200 µM), D-serine (50 µM), EGTA (450 µM), NBQX (10 µM), picrotoxin (100 µM) were purchased from Tocris Bioscience. EphrinB3-Fc (R&D Systems), a chimeric protein containing the extracellular domain of human ephrinB3 ligand fused to the C-terminal Fc region of human IgG1, was depending on the experiment clustered by a 40 min incubation at 37 °C
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Identification of ephrinB3-Fc doses to induce D-serine release in acute hippocampal slices. (A) Representative NMDAR-fEPSPs traces (on the left) are the average of 90 successive sweeps before (1) and after (2) clustered ephrinB3-Fc application. Results (on the bottom) are presented as means ± s.e.m. for NMDAR-fEPSP slopes, measured 60–90 min after clustered ephrinB3-Fc application, relative to 30 min of baseline recording. This dose response relationship led us to choose the dose of 0.5 µg/ml of ephrinB3-Fc. (B, C) The bar graphs represent the level of D-serine released and the ratio of D-serine /glycine measured in the extracellular medium of hippocampal slices using laser-induced fluorescence capillary electrophoresis. (B) Results are presented as means ± s.e.m. for changes in D-serine concentration induced by either clustered IgG1-Fc (0.27 µg/ml) or ephrinB3-Fc (0.5 µg/ml) condition relative to D-serine concentration obtained in clustered IgG1-Fc condition. Application of ephrinB3-Fc during 1 h induced an increase of D-serine concentration in the extracellular space. \* P < 0.05. Mann Whitney test, two-tailed.</figcaption></figure>

under agitation with an AffiniPure goat anti-human IgG1-Fc fragment specific antibody (1:2; Jackson ImmunoResearch). The same protocol was used for IgG1-Fc (R&D Systems).

## 2.11. Statistical analysis

Statistical differences were established at P < 0.05. Graphpad Prism software was used for statistical analysis. Data were expressed as mean ± s.e.m. Statistical test used for each experiment are detailed in each figure legends.

# 3. Results

## 3.1. Activation of EphB3 receptors increases synaptic NMDAR activity

The activation of EphB3 receptors depends on their level of multi-
merization with their ligand ephrinB3 (ephrinB3-Fc) (Blits-Huizinga
et al., 2004; Davis et al., 1994; Pasquale, 2005; Schaupp et al., 2014).
Therefore, before its bath application, the ligand ephrinB3-Fc was
clustered using an anti-IgG1 antibody which recognized the Fc part of
the ephrinB3-Fc ligand (Davis et al., 1994). We then applied increasing
doses of ephrinB3-Fc chimeras clustered while monitoring extracellular
NMDAR-field excitatory post-synaptic potentials (NMDAR-fEPSPs) in
the presence of picrotoxin (100 µM) and a low concentration of Mg²⁺
(0.2 mM) in the bathing solution. At 0.5 µg/ml, we observed a significant
increase of NMDARs responses reaching a plateau with higher doses of
the ephrinB3-Fc (124.86 ± 5.25 %, n = 10; Fig. 1A). Interestingly, the
maximal increase observed with EphB3 receptors stimulation was
similar in range (20–40 %) to that usually observed when synaptic
NMDARs are exposed to exogenous co-agonists like D-serine or glycine,
due to the fact that the level of occupancy of the NMDAR co-agonist
binding site is not fully saturated in this region (Martina et al., 2003;
Papouin et al., 2012; Robin et al., 2018).

To confirm that stimulation of EphB3 receptors lead to an increase of extracellular D-serine level (Zhuang et al., 2010), acute hippocampal slices were incubated during 1 h with either clustered IgG1-Fc (0.27 µg/ml; control condition; concentration equivalent to the one used for ephrinB3-Fc as ephrinB3-Fc has a lower molecular weight) or clustered ephrinB3-Fc (0.5 µg/ml). Then, extracellular D-serine levels were measured in acute hippocampal slices using laser-induced fluorescence capillary electrophoresis (CE-LIF). In agreement with previous reports (Zhuang et al., 2010), the stimulation of EphB3 receptors with clustered ephrinB3-Fc induced an increase of extracellular D-serine level (IgG1-Fc, 100.0 ± 10.82 %, n = 7; ephrinB3-Fc, 141.4 ± 9.03 %, n = 6; P = 0.0140; Fig. 1B).

We then decided to evaluate the impact of EphB3 receptor activation on synaptic D-serine availability and therefore on the activity of synaptic NMDARs. To this end we monitored the level of occupancy of synaptic NMDAR co-agonist-binding site by adding D-serine (50 µM) after application of either clustered IgG1-Fc or ephrinB3-Fc (Fig. 2A-C). Interestingly, bath application of D-serine had a much smaller effect on synaptic NMDAR activity when ephrinB3-Fc was applied beforehand (IgG1-Fc condition, 127.1 ± 5.69 %, n = 8; ephrinB3-Fc condition, 109.9 ± 3.37 %, n = 10; P = 0.0155; Fig. 2A-C and Fig. S1A-C), indicative of an occlusion between EphB3 receptor stimulation and D-serine application. This result suggests that the two effects share a common mode of action at CA3-CA1 synapses. Importantly, the global increase of synaptic NMDAR activity induced by the chimeras and D-serine was the same in both conditions (IgG1-Fc condition, 134.3 ± 7.47 %, n = 8; ephrinB3-Fc condition, 137.5 ± 8.02 %, n = 10; P = 0.9654; Fig. 2A). To confirm that the increase of NMDAR activity following EphB3 receptors stimulation was solely due to an increase of synaptic D-serine availability, NMDAR co-agonist binding sites were first saturated with D-serine (50 µM) before EphB3 receptors were stimulated using clustered ephrinB3-Fc. In agreement with our hypothesis, clustered ephrinB3-Fc application had no longer an impact on NMDARs activity under these conditions (D-serine condition, 133.3 ± 6.1, n = 9; ephrinB3-Fc condition, 132.5 ± 7.53; P = 0.9102; Fig. 2E, Fig. S1D-E).

## 3.2. EphB3 receptors control synaptic NMDAR activity through astrocytic Ca²⁺ signaling

We next assessed whether the increase of D-serine availability in the synapse following EphB3 receptors stimulation was astrocyte-dependent. As D-serine release is Ca²⁺-dependent, to address this, we first patched individual astrocytes with a pipette containing a Ca²⁺-clamp solution known to constrain free astrocytic Ca²⁺ (Henneberger et al., 2010) while monitoring NMDAR activity at neighboring synapses through astrocytic-fEPSPs (a-fEPSPs), as described by Henneberger and colleagues (Henneberger et al., 2010; Henneberger and Rusakov, 2012). Indeed, as reported before, the Ca²⁺-clamp solution (see material and methods; Henneberger et al., 2010) allows to clamp Ca²⁺ concentration to 50-80 nM, inhibiting activity induced increase in Ca²⁺ concentration. Astrocytes were identified morphologically (Fig. 3A) and biophysically by their low input resistance, hyperpolarized resting membrane potential, linear current-voltage relationship (Fig. 3B) and absence of action potential (Fig. 3C). In the presence of AMPAR blocker (NBQX, 10 µM) and low Mg²⁺ (0.2 mM), the field potential recorded through the astrocyte patch pipette was mediated by NMDARs, glutamate transporter (GLT-1) and long-lasting inward potassium currents (Fig. 3D)
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** EphB3 receptor activation leads to an increase of synaptic NMDARs activity. (A) Representative NMDAR-fEPSPs traces are the average of 90 successive sweeps before (1) and after (2) bath applications of either clustered IgG1-Fc (0.27 µg/ml) or ephrinB3-Fc (0.5 µg/ml) and then (3) D-serine (50 µM). While IgG1-Fc had no impact, ephrinB3-Fc induced an increase of NMDARs activity through an increase of the co-agonist binding site occupancy. (B), (C) Bar graphs summarizing results presented in (A). \* P < 0.05; \*\* P < 0.01. Mann Whitney test, two-tailed. (B) The bar graph shows the effect of clustered chimeras on synaptic NMDAR responses. Results are presented as means ± s.e.m. for NMDAR-fEPSP slopes measured after clustered chimera application (60–90 min) relative to baseline (–30–0 min). (C) Effect of D-serine on synaptic NMDAR responses in clustered chimera condition. Results are presented as means ± s.e.m. for NMDAR-fEPSP slopes measured after D-serine application (100–130 min) relative to clustered chimera condition (60–90 min). (D) Representative NMDAR-fEPSPs traces before (1) and during (2) bath applications of first D-serine and then (3) clustered ephrinB3-Fc. This occlusion experiment, represented on the plot, shows the specificity of action of ephrinB3-Fc on the release of D-serine. EphrinB3-Fc had no additional effect of the co-agonist binding site occupancy as it was first fully saturated by exogenous D-serine (50 µM). (E) Each connected circle represents paired mean NMDAR-fEPSP slope, for each individual experiment averaged in (D) measured in D-serine condition before (10–40 min) and after clustered ephrinB3-Fc application (100–130 min), relative to baseline (–30–0 min). n.s. (P > 0.05). Wilcoxon signed-rank test.</figcaption></figure>

(Henneberger et al., 2012; Sibille et al., 2014). At the end of the experiment, signals obtained in the presence of NMDAR antagonist (AP5, 50 µM; Fig. 3D) were subtracted to obtain a NMDAR-fEPSPs. Importantly, a-NMDAR-fEPSPs slopes monitored through an astrocyte patch pipette fitted significantly with the slope of NMDAR-fEPSPs recorded with a standard extracellular electrode close to the soma of the patched astrocyte (r = 0.9763, n = 17; P < 0.0001; Figs. 3A and 3E).

While clustered ephrinB3-Fc induced an increase of the activity of synaptic NMDARs, this effect was no longer observed in Ca²⁺-clamp condition (control condition, 124.7 ± 6.70 %, n = 6; Ca²⁺-clamp condition, 96.08 ± 4.99 %, n = 6; P = 0.0087; Fig. 3F). This finding indicates that EphB3 receptors control NMDAR activity through the release of D-serine from astrocytes.

### 3.3. EphB3 receptors endogenously regulate NMDAR activity

In view of the effects of EphB3 receptors stimulation onto NMDAR activity, we next investigated whether a similar regulation was

prevailing endogenously. To tackle this issue, endogenous EphB3-ephrinB3 interaction was inhibited with exogenous unclustered ephrinB3-Fc, an experimental process commonly used to inhibit EphB3 tonic activation (Contractor et al., 2002; Lawrenson et al., 2022; Murai et al., 2003; Stein et al., 1998). We observed that applications of unclustered ephrinB3-Fc (2.0 µg/ml) led to an impairment of NMDAR activity (uncl. IgG1-Fc, 1.08 µg/ml, 104.3 ± 4.81 %, n = 8; uncl. ephrinB3-Fc, 81.33 ± 5.33 %, n = 7; P = 0.0205; Fig. 4A and B). Interestingly, applying D-serine yielded a larger increase in the presence of unclustered ephrin B3, increasing NMDAR response to levels similar to those obtained under control conditions (uncl. IgG1-Fc condition, 123.2 ± 7.46 %, n = 8; uncl. ephrinB3-Fc condition, 134.7 ± 12.11 %, n = 7; P = 0.6943; Fig. 4A).

These data indicate that the NMDAR impairment observed when inhibiting EphB3 receptors was caused by a reduction in co-agonist supply. This was further supported by the fact that bath-application of D-serine completely occluded the effect of unclustered ephrinB3-Fc (D-serine condition, 125.5 ± 5.53, n = 6; uncl. ephrinB3-Fc condition,
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** EphB3 receptors control synaptic NMDAR activity astrocytic Ca²⁺ signaling. (A) Overview image of the experimental arrangement. In the *stratum radiatum* of the CA1 region, whole-cell patch-clamp recording of an astrocyte (patch-clamp pipette, IP) was realized at the same time as extracellular recordings using an extracellular pipette (EP) placed near the soma. (B) Linear current-voltage relationship in voltage-clamp configuration. (C) Steps of voltage induced by square pulses of currents (800 pA steps) in current-clamp configuration shows that no action potential was induced. (D) Representative astrocytic-NMDAR-fEPSPs (a-NMDAR-fEPSPs) during successive bath application of (1) picrotoxin (100 µM) + NBQX (10 µM), (2) D-serine (50 µM), (3) AP5 (50 µM) and (4) DL-TBOA (200 µM). (E) Correlation between NMDAR-fEPSP slope and a-NMDAR-fEPSP slope. Representative simultaneous NMDAR-fEPSP and a-NMDAR-fEPSP at different percentages of the maximum response. Results are presented as means for slopes relative to the highest slope (set at 100 %) of each animal. a-NMDAR-fEPSPs fitted significantly with the slope of NMDAR-fEPSPs recorded with a standard extracellular electrode closed to the soma. n = 17 slopes, 4 mice. r = 0.9763; P < 0.0001. Spearman correlation test. (F) Representative a-NMDAR-fEPSPs traces are the average of 30 successive sweeps before (1) and after (2) clustered ephrinB3-Fc (0.5 µg/ml) application. Bar graph illustrates clustered ephrinB3-Fc effect on synaptic NMDAR responses recorded through an astrocyte loaded with control or Ca²⁺-clamp solutions (in mM: 0.2 BAPTA, 0.45 EGTA and 0.14 Ca²⁺). Results are presented as means ± s.e.m. for a-NMDAR-fEPSP slopes, measured 50–60 min after ephrinB3-Fc application, relative to 10 min of baseline recording. \*\* P < 0.01. Mann Whitney test, two-tailed.</figcaption></figure>

124.6 ± 8.75; P > 0.9999; Figs. 4D and 4E).

Importantly, astrocytes are involved not only in the control of synaptic D-serine availability but also play a role in glutamate homeostasis (Murphy-Royal et al., 2015; Oliet et al., 2001). We thus tested whether our experimental manipulation of EphB3 receptors affected excitatory transmission through mechanisms other than D-serine supply. Neither clustered nor unclustered ephrinB3-Fc application modified synaptic AMPAR-dependent glutamatergic transmission (ephrinB3-Fc, 99.63 ± 5.59 %, n = 5; P = 0.8125; uncl. ephrinB3-Fc, 102.4 ± 8.71 %, n = 5; P > 0.9999; Fig. S2A and S2B). Paired-pulse ratios (PPRs), indicators of the presynaptic release probability, were not affected neither by clustered (before ephrinB3-Fc, 1.54 ± 0.06 %; in ephrinB3-Fc, 1.58 ± 0.06 %; n = 5; P = 0.0625; Fig. S2C) nor unclustered ephrinB3-Fc application (before uncl. ephrinB3-Fc, 1.63 ± 0.03 %; in uncl. ephrinB3-Fc, 1.71 ± 0.04 %; n = 5; P > 0.0625; Fig. S2D). Consistent with these results, the extracellular glutamate levels were similar in control or clustered ephrinB3-Fc conditions (IgG1-Fc, 100.0 ± 11.77 %, n = 5; ephrinB3-Fc, 111.4 ± 22.58 %, n = 5; P = 0.8413; Fig. S2E). Altogether, our data suggest that glutamatergic AMPAR-mediated transmission is not affected by ephrinB3-Fc application, thereby confirming the specific control of D-serine availability and NMDAR transmission by EphB3-ephrinB3 interactions.

### 3.4. Astrocytic EphB3 receptors regulate LTP induction and novel object recognition memory

As manipulating EphB3 receptors regulates the activity of synaptic NMDARs, we investigated whether it has an impact on LTP induction at

CA3-CA1 synapses. To answer this question, hippocampal slices were incubated for 1 h with either unclustered IgG1-Fc or unclustered ephrinB3-Fc before inducing LTP using a high frequency stimulation protocol (HFS; 3 × 100 Hz, 1 sec duration, 20 sec interval). Whereas LTP was readily obtained in the presence of unclustered IgG1-Fc, it was impaired significantly under conditions where endogenous EphB3-ephrinB3 interaction was inhibited using unclustered ephrinB3-Fc (uncl. IgG1-Fc, 138.7 ± 7.12 %, n = 6; uncl. ephrinB3-Fc, 106.7 ± 5.84 %, n = 7; P = 0.0082; Fig. 5A, C). Post-tetanic potentiation (PTP), measured 1 min after the tetanus was unaffected (Fig. 5B). Strikingly, LTP was fully rescued by exogenous D-serine (50 µM) (uncl. IgG1-Fc, 138.7 ± 8.58 %, n = 6; uncl. ephrinB3-Fc, 145.7 ± 7.29 %, n = 6; P = 0.3095; Figs. 5D, 5F), indicating that EphB3-ephrinB3 signaling controls LTP induction through the regulation of synaptic D-serine availability, in agreement with the effects observed on NMDAR responses.

To confirm that EphB3 receptors responsible for regulating the D-serine supply are indeed astrocytic, we took advantage of a viral approach to knock down EphB3 receptors in astrocytes using short hairpin (sh) RNA interference. To do so, 5 weeks before performing electrophysiological experiments, AAV2.8 viruses allowing the expression of microRNA (mir30) based-shRNA sequence targeting EphB3 mRNA (shEphB3) together with DsRed under the astrocytic GFAP promoter were transfected in the CA1 area of the hippocampus (Fig. 6, Fig. S3 and Fig. S4, see material and methods). A control group was transfected with an AAV2.8 allowing either the expression of GFP only in astrocytes (CTL group; Fig. 6 and Fig. S3, see material and methods) or a short hairpin RNA targeting a non-coding sequence and DsRed
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Endogenous EphB3-ephrinB3 interaction regulates synaptic *D*-serine availability and thus synaptic NMDAR activity. (A) Representative NMDAR-fEPSPs traces are the average of 60 successive sweeps before (1) and after (2) bath applications of unclustered IgG1-Fc (1.08 µg/ml) or ephrinB3-Fc (2.0 µg/ml) and then (3) *D*-serine (50 µM). The inhibition of endogenous EphB3-ephrinB3 interaction by unclustered ephrinB3-Fc induced a decrease of the activity of NMDARs. This effect was rescued by exogenous application of *D*-serine (50 µM).(B), (C) Bar graphs summarize results presented in (A). \* P < 0.05. Mann Whitney test, two-tailed. (B) The bar graph represents the effect of unclustered chimeras on synaptic NMDAR activity. Results are presented as means ± s.e.m. for NMDAR-fEPSP slopes measured after unclustered chimera application (70–90 min) relative to baseline (-20–0 min). (C) This bar graph shows the effect of *D*-serine on synaptic NMDAR activity in unclustered chimera condition. Results are presented as means ± s.e.m. for NMDAR-fEPSP slopes measured after *D*-serine application (110–130 min) relative to unclustered chimera condition (70–90 min).(D) Representative NMDAR-fEPSPs traces are the average of 60 successive sweeps before (1) and during (2) bath applications of *D*-serine and then (3) unclustered ephrinB3-Fc. The effect of unclustered ephrinB3-Fc was occluded by previous application of exogenous *D*-serine (50 µM). (E) Each connected circle represents paired mean NMDAR-fEPSP slope, for each individual experiment averaged in (D) measured in *D*-serine condition before (20–40 min) and after unclustered ephrinB3-Fc application (110–130 min), relative to baseline (-20–0 min). n.s. (P > 0.05). Wilcoxon signed-rank test.</figcaption></figure>

(shCTL group, Fig. S4A-B, see material and methods).

Then, we assessed the consequence of knocking down EphB3 receptors in astrocytes, by monitoring tetanus-induced LTP at CA3-CA1 synapses in acute slices 5 weeks after hippocampal transfection. As observed earlier with exogenous application of unclustered ephrinB3-Fc (Fig. 5), while PTP was not affected (Fig. 6B), tetanic LTP was impaired significantly in the shEphB3 group of animals (CTL, 161.7 ± 5.85 %, n = 5; shEphB3, 101.3 ± 7.98 %, n = 7; P = 0.0025; Fig. 6A, C). Importantly, LTP was fully restored in the presence of exogenous *D*-serine (50 µM) in the bathing solution (CTL, 163.9 ± 6.0 %, n = 7; shEphB3, 207.7 ± 16.35 %, n = 6; P = 0.0221; Fig. 6D, F).

Finally, we took advantage of this cell-specific knock-down to investigate the role of astrocytic EphB3 receptors in regulating *D*-serine supply and NMDAR activity on memory. Similar to what we did in a previous study (Robin et al., 2018), we used long-term novel object recognition memory task in a L-maze to test our animals. The memory performance was largely impaired in the shEphB3 group in comparison

to the control group (CTL, 0.418 ± 0.04, N = 6; shEphB3, 0.05 ± 0.021, N = 6; P < 0.001; Fig. 7). Most interestingly, systemic injection of *D*-serine (i.p., 50 mg/kg; Robin et al., 2018) immediately after acquisition fully rescued memory performances of shEphB3 mice (CTL, 0.364 ± 0.08, N = 6; shEphB3, 0.357 ± 0.05, N = 6; P > 0.05; Fig. 7).

## 4. Discussion

In the present work we showed that disrupting astrocytic EphB3 receptors impaired NMDAR functions. Our data reveals that EphB3 receptors regulate *D*-serine availability at CA3-CA1 synapses. This in turn has functional consequences on NMDAR activity and synaptic plasticity, making astrocytic EphB3 receptors important protagonists in hippocampal-based memory processes

EphB3 receptors control *D*-serine availability at excitatory synapses

While different receptors have been proposed to be involved in the release of *D*-serine (Fernández-Moncada et al., 2024; Mothet et al., 2005;
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** EphB3-ephrinB3 interaction regulates LTP induction. (A), (D), Representative fEPSPs traces are the average of 30 successive sweeps before (1) and after (2) high frequency stimulation (HFS; 3 ×100 Hz, 1 sec, 20 sec interval; arrows). Slices were incubating 1 h with either unclustered IgG1-Fc (1.08 µg/ml) or ephrinB3-Fc (2.0 µg/ml), and 20 min with (D) or without (A) d-serine (50 µM), before HFS.(B), (E) Bar graphs representing the post-tetanic potentiation (PTP) measured the first minute after the induction. Results are presented as means ± s.e.m. for fEPSP slopes measured just after HFS (0–1 min) relative to baseline (-10–0 min). While the PTP was not altered (B), the LTP (A, C) was impaired when the endogenous interaction of EphB3 receptor with its endogenous ligand was decreased using unclustered ephrinB3-Fc.(C), (F) Bar graph summarizing the potentiation of synaptic transmission in (A) and (D). Results are presented as means ± s.e.m. for fEPSP slopes measured after HFS (50–60 min) relative to baseline (-10–0 min). ** P < 0.01. Mann Whitney test, two-tailed.**</figcaption></figure>

Robin et al., 2018; Schell and Molliver, 1995; Sherwood et al., 2017; Sherwood et al., 2021; Shigetomi et al., 2013; Yang et al., 2003), we here show that an astrocytic CAM protein, the EphB3 receptor, is involved in this process. During electrophysiological recordings, we demonstrated that stimulating EphB3 receptors leads to synaptic release of d-serine, whereas inhibiting EphB3-ephrinB3 interactions impairs d-serine availability at CA3-CA1 synapses. These results are in full agreement with a role for EphB3 receptors in regulating synaptic NMDARs. Considering that d-serine is the endogenous co-agonist of synaptic NMDARs at CA3-CA1 synapses in adult mice (Ferreira et al., 2017; Papouin et al., 2012), but does not play such a role at extrasynaptic sites (Papouin et al., 2012), the question arises whether there is a mechanism allowing the localized supply of d-serine specifically at synapses. Interestingly, the group of Dalva (Hruska et al., 2015) showed that the ligand of the EphB3 receptor, ephrinB3, which is expressed at postsynaptic sites, controls the

localization and stabilization of PSD95. Thus, we can hypothesize that through the interaction with its ligand, EphB3 receptors would allow the appropriate localization at the vicinity of synaptic sites of the machinery involved in d-serine supply.

It is important to notice that a modification of the activity of NMDARs can occur through other mechanisms like a modification of their biophysical properties (Paoletti, 2011). Indeed, by interacting with NMDARs, neuronal EphB2 receptors regulate the level of Ca²⁺ influx (Dalva et al., 2000; Grunwald et al., 2001; Nolt et al., 2011; Takasu et al., 2002). However, this does not seem to account for our findings since EphB3 activation did not have any further effect on NMDAR activity once the co-agonist-binding sites were fully occupied by exogenous d-serine. Moreover, EphB3 activation did not have any effect either on glutamate level, presynaptic release probability or glutamatergic AMPAR-mediated transmission indicating that it did not have any
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Astrocytic EphB3 receptors are important for LTP induction. (A), Representative fEPSPs traces are the average of 20 successive sweeps before and after high frequency stimulation (HFS; 3 × 100 Hz, 1 sec, 20 sec interval; arrows). Plots showing fEPSPs slopes before and after HFS. (B), (E) Bar graphs representing the post-tetanic potentiation (PTP) measured the first minute after the induction. Results are presented as means ± s.e.m. for fEPSP slopes measured just after HFS (0–1 min) relative to baseline (-10–0 min). (C) Bar graph showing the potentiation of synaptic transmission. Results are presented as mean ± s.e.m. for fEPSPs slopes measured after HFS (50–60 min) relative to baseline (-10–0 min). While the PTP was not modified (B), the LTP was impaired when the EphB3 receptors were exclusively deleted in astrocytes using shEphB3 virus. \*\* P < 0.01. Mann Whitney test, two-tailed. (D) Representative fEPSPs traces are the average of 20 successive sweeps before and after HFS. Plots showing fEPSPs slopes before and after HFS. Slices were incubated with D-serine (50 µM) 20 min before and during all the recording. (F) Bar graph showing the potentiation of synaptic transmission. Results are presented as mean ± s.e.m. for fEPSP slopes measured after HFS (50–60 min) relative to baseline (-10–0 min). The LTP was rescued when exogenous D-serine was applied in the bath before HFS. \* P < 0.05. Mann Whitney test, two-tailed.</figcaption></figure>

impact on the localization of the astrocytic process in regards to the synapse. The absence of effects on AMPAR transmission is in accordance with results obtained in knock down animals for EphB3 (Perez et al., 2016; Zhuang et al., 2010) or ephrinB3 (Grunwald et al., 2004; Rodenas-Ruano et al., 2006). To summarize, our data indicate that the control of EphB3 receptors on synaptic NMDAR activity is mediated through D-serine supply.

EphB3 receptors involved in this process are astrocytic

The release of D-serine by astrocytes is a Ca²⁺-dependent process (Henneberger et al., 2010; Shigetomi et al., 2013). Thus, to confirm that EphB3 receptors involved in this process are expressed by astrocytes we decided to occlude the effect of EphB3 receptor activation by first

clamping the level of Ca²⁺ specifically in astrocytes in order to antagonize D-serine release (Henneberger et al., 2010). Under these conditions, the activation of EphB3 receptors no longer affected the activity of synaptic NMDA receptors, as expected from the involvement of astrocytic D-serine. Importantly, knocking down EphB3 receptors specifically in astrocytes, using a viral approach to induce the expression of a short hairpin RNA sequence targeting EphB3 mRNA (shEphB3) in astrocytes, impaired LTP induction as well as novel object recognition memory. This impairment was specifically due to a decrease of D-serine availability as they were both rescued by exogenous D-serine.

However, the precise molecular pathway through which EphB3 receptors regulate the release of D-serine remains unclear. It is interesting
<figure><img src="image_9.png" /><figcaption>**Fig. 7.** Astrocytic EphB3 receptors are important for novel object recognition memory. (A) Schematic of AAV2.8 transfection in the CA1 area of either the control virus (CTL group) or the virus allowing the expression of the shEphB3 in astrocytes (shEphB3 group) 5 weeks before the novel object recognition memory task. The task consisted of 3 sequential daily trials of 9 min each. During the habituation session (day 1, trial 1), the mouse freely explored the arms in the absence of any objects. During the acquisition session (day 2, trial 2) the mouse was placed in the presence of two identical objects positioned at the extremities of each arm. Right after this acquisition session, the mouse was injected intraperitoneally with either *d*-serine (50 mg/kg in saline solution) or saline as vehicle control. The memory test occurred 24 h later (day 3, trial 3). (B) Exploration time of arm A vs arm B of the NOR apparatus during training. Results are presented as means ± s.e.m. Two-Way ANOVA, Sidak's multiple comparisons test. (C) Exploration time of familiar vs novel objects in the NOR test. Results are presented as means ± s.e.m. **** P < 0.0001. Two-Way ANOVA, Sidak's multiple comparisons test. (D) Bar graph illustrating the total exploration time of mice. Results are presented as means ± s.e.m. Two-Way ANOVA. (E) Bar graph illustrating the memory performance of mice in the novel object recognition memory task assessed by the discrimination index calculated as the difference between the time spent exploring the novel (TN) and the familiar object (TF) divided by the total exploration time (TN+TF). Results are presented as means ± s.e.m. ** P < 0.01; *** P < 0.001. Two-Way ANOVA, Sidak's multiple comparisons test.</figcaption></figure>

to note that Eph receptors activation can lead to activation of the
phospholipase C gamma 1 (PLCγ1) (Zhou et al., 2007). As PLCγ1 is
expressed by astrocytes, we can hypothesize that activation of EphB3
receptors could control locally the level of Ca²⁺. In addition, activation
of this receptor could regulate the level of *d*-serine synthesis from
*l*-serine. Indeed, data from the group of Liebl (Zhuang et al., 2010, 2011)
suggest strongly that activation of astrocytic EphB3 receptors control the
level of *d*-serine through a decrease of PKC alpha phosphorylation and
an increase of the association of the serine racemase with PICK1.
Intriguingly, our recent data indicate that short-term activation of
non-mitochondrial associated astrocytic CB1 receptors require the ac-
tivity of PKC for the production and release of lactate (Ferna-
dez-Moncada et al., 2024). Once released, lactate activates the HCAR1
signaling, switching glycolysis towards serine production, and ulti-
mately providing the NMDAR activity necessary for physiological
memory processing (Fernandez-Moncada et al., 2024). Thus, both

astrocytic CB1 and EphB3 receptors control synaptic NMDAR functions. Future studies will investigate the potential mechanistic interactions between these two signaling systems in determining neuron-to-astrocyte crosstalk leading to synaptic *d*-serine availability. Importantly, while *d*-serine can be synthesized by both astrocytes and neurons (Coyle et al., 2020; Sherwood et al., 2021), the present work is an additional piece of evidence demonstrating that specific manipulations of astrocytes lead to impaired NMDAR co-agonist release. While *d*-serine can be synthesized and then released by astrocytes, it has been proposed that *d*-serine synthesized in neurons is transported to astrocytes where it could be released to act locally at synaptic sites (Abreu et al., 2023). As reported before (Papouin et al., 2017), it was shown in this study (Abreu et al., 2023) that the inhibition of the SNARE-dependent exocytosis from astrocytes altered LTP induction, a process that was rescued by *d*-serine but not by *l*-serine. This argues strongly in favor of astrocytes as the site of synaptic *d*-serine release, establishing the contribution of astrocytic
D-serine in governing LTP (Abreu et al., 2023).

In the present work, all the recordings were performed at CA3-CA1 synapses within the *stratum radiatum* at mid-range within the layer. While it has been reported that in the mouse hippocampus astrocytes are heterogenous in term of morphology across layers (Viana et al., 2023) and with distinct cell interactions (Refaeli et al., 2021), we did not observed variability in our experiments that would suggest such an astrocytic heterogeneity in the context of D-serine supply.

Astrocytic EphB3 receptors control LTP induction

As astrocytes regulate the level of NMDAR activity through astrocytic EphB3 receptors activation, they also regulate LTP induction which depends on NMDAR at CA3-CA1 synapses. Indeed, our data show that while LTP was impaired when EphB3 receptors were inhibited, it was rescued by exogenous D-serine. Previous studies have shown that EphB3-ephrinB3 interaction is crucial for LTP induction. Indeed, LTP was impaired in mice in which EphB3 receptors (Perez et al., 2016; Zhuang et al., 2010) or ephrinB3 ligand (Grunwald et al., 2004; Rodenas-Ruano et al., 2006) have been completely knocked out, in all cell types. However, the removal of the intracellular part of ephrinB3 ligand led to normal LTP (Rodenas-Ruano et al., 2006). Consistent with previous findings establishing that stimulation of EphB3 receptors on astrocyte induces the release of D-serine (Zhuang et al., 2010), we here show that such control of D-serine availability by astrocytic EphB3 receptors at CA3-CA1 synapses is essential for enabling activation of synaptic NMDARs, thereby allowing a proper LTP induction and its associated novel object recognition memory.

Even though our data corroborate many studies (Grunwald et al., 2004; Perez et al., 2016; Rodenas-Ruano et al., 2006; Zhuang et al., 2010), 2 other groups have failed to report changes in LTP in the absence of ephrinB3 (Antion et al., 2010; Armstrong et al., 2006). Interestingly, while all the negative studies were carried out on juvenile animals, those describing an impaired LTP were performed on older mice. This could account for the discrepancy since it has been shown that there is a developmental switch in the nature of the co-agonist endogenously gating synaptic NMDARs from glycine to D-serine (Ferreira et al., 2017). Thus, the role of EphB3-ephrinB3 interaction in the induction of LTP might depend on the developmental stage of the animal and could only prevail in adult mice under conditions where D-serine, and not glycine, is the endogenous co-agonist of synaptic NMDARs. Interestingly, ephrinB3 RNA expression level has been shown to increase strongly in the CA1 area at the end of the development (Liebl et al., 2003). We can thus hypothesize that such an increase leads to a reinforcement of the astrocytic EphB3-neuronal ephrinB3 interaction, improving then the astrocytic control of synaptic D-serine availability, NMDAR transmission, LTP and memory.

In conclusion, astrocytic EphB3 receptors control D-serine availability at CA3-CA1 synapses. By doing so, EphB3-ephrinB3 interactions are of paramount importance for the astrocytic-neuronal crosstalk that regulates NMDAR activity. As such, investigating these cell-to-cell physical interactions might provide new insights in our understanding of the many physiological and pathological processes that depends on NMDARs.

## Authors contribution

V.C. L. performed electrophysiological experiments using chimeras and wrote the manuscript; S.M. performed electrophysiological experiments on transfected animals and wrote the manuscript; A.A., M.M., A.J. helped with experiments; A.C. performed experiments for the generation of the viral approach; F.J.-K. performed immunohistochemistry experiments; I.B. and I.M. performed laser-induced fluorescence capillary electrophoresis experiments; M.M. and T. L.L. performed laser captured microdissection and quantitative real-time PCR; S.M. and E.A. helped with experiments; G.M. helped with viral experiments; L.B. designed the shRNA, viral approach and performed behavioral experiments; S.H. R.O and A.P. supervised the work of V.L.; S.H.R.O. and A.P. wrote the

manuscript; A.P. conceived the study and wrote the manuscript; all authors edited and approved the manuscript.

## CRediT authorship contribution statement

**Langlais Valentin Clément:** Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. **Mountadem Sarah:** Writing – original draft, Investigation, Formal analysis, Data curation. **Benazzouz Ines:** Methodology. **Amadio Aurélie:** Investigation. **Matos Marco:** Investigation. **Jourdes Aurélie:** Investigation. **Cannich Astrid:** Methodology, Investigation. **Julia-Kalajzic Francisca:** Investigation. **Belluomo Ilaria:** Methodology. **Matias Isabelle:** Methodology. **Maitre Marlène:** Methodology. **Lesté-Lasserre Thierry:** Methodology. **Marais Sébastien:** Investigation. **Avignone Elena:** Investigation. **Marsicano Giovanni:** Methodology. **Bellocchio Luigi:** Investigation, Data curation, Conceptualization. **Oliet Stéphane Henri, Richard:** Writing – original draft, Supervision. **Panatier Aude:** Writing – review & editing, Writing – original draft, Supervision, Data curation, Conceptualization.

## Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

We would like to thank all the personnel of the Animal Facilities of the Neurocentre Magendie for mouse care as well as all the personnel of PUMA (MAgendie Unified Platforms). The microscopy was done in the Bordeaux Imaging Center, a service unit of the CNRS-INSERM and Bordeaux University, member of the national infrastructure France BioImaging supported by the French National Research Agency (ANR-10-INBS-04). We thank all past and present members of Oliet's lab for useful discussions. This study was funded by INSERM (to A.P., S.H.R.O., G.M. and L.B.); CNRS (to A.P. and S.H.R.O.); the Agence Nationale de la Recherche (ASTRODOM, AAP JCJC 2016 to A.P.; Excigly, ANR AAPG2017 to S.H.R.O.; Astrocom AAPG2019 to A.P.); Fondation pour la Recherche Médicale (V.C.L); French Ministry of Higher Education and Research (V.C.L). and the French government in the framework of the University of Bordeaux's IdEx "Investments for the Future" program / GPR BRAIN_2030).

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.pneurobio.2025.102747.

## Data availability

Data will be made available on request.

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