Impaired metabotropic glutamate type 5 receptor signaling in the dorsal striatum of the R451C-neuroligin 3 mouse model of Autism Spectrum Disorder

doi:10.21203/rs.3.rs-4893764/v1

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Impaired metabotropic glutamate type 5 receptor signaling in the dorsal striatum of the R451C-neuroligin 3 mouse model of Autism Spectrum Disorder

https://doi.org/10.21203/rs.3.rs-4893764/v1

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Background

Human genetics indicates enrichment of synaptic pathway-related mutations in Autism Spectrum Disorder (ASD). Accordingly, several preclinical studies have reported synaptic alterations in different brain areas of relevant ASD mouse models. In particular, we previously showed that corticostriatal long-term synaptic depression is impaired in the dorsal striatum of mice carrying the ASD-associated R451C mutation in the neuroligin3 gene.

Methods

We used behavioral, proteomic, biochemical, and electrophysiological approaches to explore the dorsal striatum-dependent functions in the R451C-neuroligin3 mouse model of ASD.

Results

A detailed behavioral analysis confirmed striatum-dependent alterations in these mice. We further explored the corticostriatal synaptic function, disclosing modifications of the glutamatergic postsynaptic density protein composition, which functionally result in the impairment of different forms of corticostriatal synaptic plasticity, namely activity-dependent long-term depression and potentiation, and group I metabotropic glutamate receptor-dependent synaptic depression. We also found reduced protein expression levels of type 5 metabotropic glutamate receptor at striatal synapses, which likely preclude the expression of long-term potentiation and depression by preventing the potentiation of NMDA receptor-mediated currents and a sufficient generation of endocannabinoids, respectively.

Conclusions

Overall, our findings point to a significant impairment of type 5 metabotropic glutamate receptor signaling, affecting the dorsal striatum function, which underlies specific autism-relevant behaviors in R451C-neuroligin3 mice.

Behavior

proteomics

electrophysiology

glutamate metabotropic receptors

corticostriatal synapses

Genetic studies indicate a complex and heterogeneous etiology of Autism Spectrum Disorder (ASD). Among commonly affected signaling pathways, those involving synapse formation and regulation of synaptic transmission have been recurrently reported. In particular, over the past 15 years, accumulating evidence has implicated the abnormal expression, signaling, and function of group I metabotropic glutamate (mGluI) receptors in autism [1–4] and autism-related disorders, such as Fragile X syndrome (FXS) [5–7], obsessive-compulsive disorder (OCD) [8], and intellectual disability (ID) [9, 10]. MGluI receptors include mGlu1 and mGlu5 subtypes, which are key players in glutamate-mediated excitatory transmission and overall brain function, as their activity modulates neuronal excitability, intracellular Ca²⁺ signaling, protein synthesis, and dendritic spine maturation [11]. These metabotropic receptors mediate the action of glutamate either via Gq/11 proteins, phospholipase C, protein kinase C (PKC), diacylglycerol, fostering Ca²⁺ mobilization from internal stores, and finally the MAPK/ERK pathway, or via a Gq/11-independent pathway involving phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR) [12]. Notably, mGlu5 receptors are expressed in the brain from early developmental stages [13, 14]. The high degree of sequence homology and common signaling mechanisms between mGlu1 and mGlu5 receptors led to the hypothesis that they had redundant functions, but subsequent evidence suggested that these receptors either had also distinct functions [15–19], or cross-talked, acting either cooperatively [20] or antagonistically [21, 22]. Although mGlu1 and mGlu5 receptors can be also found at presynaptic terminals [23], in astrocytes and microglial cells [24], they are mainly localized in the perisynaptic areas of postsynaptic densities (PSDs) [25, 26], and their activation can lead to cell depolarization and enhanced neuronal excitability. Notably, mGluI receptors play important roles in the expression of long-lasting forms of synaptic plasticity, long-term potentiation and depression (LTP and LTD, respectively) [27–33]. In particular, the expression of LTD at corticostriatal synapses depends on the activation of both cortical glutamatergic and nigral dopaminergic inputs to striatal spiny projection neurons (SPNs), leading to both depolarization and activation of mGlu and dopamine type 2 (D2Rs) receptors. This cascade of events leads to the production of endocannabinoids (eCBs), which retrogradely reach and bind presynaptic cannabinoid CB1 receptors, located on glutamatergic terminals, resulting in a long-term decrease in the probability of neurotransmitter release [34, 35]. Of note, we previously demonstrated the loss of LTD expression at corticostriatal synapses of the dorsal striatum [36] in a mouse model carrying the ASD-associated point mutation R451C in the gene encoding the synaptic adhesion protein neuroligin 3 (NL3), the R451C-neuroligin 3 (R451C-NL3) mice. Our study further showed that activation of eCB signaling was able to partially rescue LTD expression at the corticostriatal synapses of R451C-NL3 mice [36]. This observation led us to hypothesize that the origin of striatal synaptic dysfunction might lie upstream the eCB production in the LTD induction pathway. In the present study, we therefore examined the expression and function of both mGlu1 and 5 receptor subtypes, in the dorsal striatum of R451C-NL3 mice, by using proteomic, biochemical, and electrophysiology approaches. Our findings reveal an impairment of mGlu5 receptor protein expression and function at R451C-NL3 dorsal striatum synapses, and further support the involvement of these receptors in ASD pathophysiology.

Experimental Model

Studies were carried out on adult (P60-P90) R451C-NL3 male and female mice (B6;129-Nlgn3tm1Sud/J) and their wild-type (WT) littermates. All the experiments were approved by the ethics committee of IRCCS Fondazione Santa Lucia, and authorized by the Italian Ministry of Health (nr. 740/2015-PR, 492/2019-PR, and 739/2022-PR), in accordance to the Italian D. Lgs 26/2014 and the European Union Directive 2010/63/EU. All the efforts were made to minimize the number of animals and their distress and suffering. R451C-NL3 knock-in mice (KI) and their control littermates were housed in 4 per cage and maintained with food and water ad libitum, with a 12/12 h light/dark cycle and constant temperature (22 ± 2°C). For genotyping, the R451C mutation was detected after PCR amplification of the insertion site of the first loxP site in the NL3 gene (FW 5′-TGTACCAGGAATGGGAAGCAG-3′; RW 5′-GGTCAGAGCTGTCATTGTCAC-3′) from genomic DNA extracted from the mouse tail, as previously described [36].

Behavioral Assays

Two-month-old male R451C-NL3 mice and age-matched littermates were used for the behavioral analysis. After handling habituation, mice were subjected to a battery of behavioral tests as detailed below, each of which was administered once, between 8 AM and 4 PM, on different days. To explore behaviors in the R451C-NL3 animal model of ASD, we utilized the Research Domain Criteria (RDoC) framework, which aims at establishing an experimental categorization system where symptoms/domains are classified by anchoring them to the etiological and pathophysiological evidence (https://www.nimh.nih.gov/research/research-funded-by-nimh/rdoc/index.shtml).

Three-chamber social interaction test

The 3-chamber test was used to study sociability and social novelty, as previously described [37]. The experimental apparatus consisted of a rectangular plexiglass box (60 x 40 x 22 cm) divided into three identical compartments connected by two square openings. One hour prior to the test, animals underwent isolation and acclimatization to the apparatus. The experimental procedure began by placing the test mouse in the central compartment, allowing it to freely explore the three chambers for a duration of 10 minutes (habituation). Afterwards, for the Sociability Phase an inanimate object was placed in a wire cup in one of the chambers, and an age-matched stranger wild-type male mouse in a similar wire cup in the other chamber, and the test mouse was allowed to choose to interact with one or the other. After 10 minutes, the chambers were cleaned, and, for the Social Novelty phase (ten minutes duration), the stranger conspecific was reintroduced in the same chamber, and a novel unfamiliar mouse replaced the object in the first chamber. Each session was recorded using a digital video camera placed above the cage and monitored using Ethovision XT 16 (Noldus). The position of the stranger mouse was randomized between the left and right-side chambers to rule out possible chamber preferences. The animals used as strangers were habituated to the wire cup during 5 minutes before the test. Interaction was determined as the moment when the nose of the test mouse was directed towards the wire cup, at less than 2 cm of distance. The Sociability index evaluates the preference for a conspecific (S) with respect to an object (O), and is measured by the percentage of time spent in close interaction with either S or O on the total time spent interacting with S and with O. The Social novelty index evaluates preference for the novel (N) with respect to the familiar (F) conspecific, measured by the percentage of time spent in close interaction with either N or F on the total time spent interacting with N and with F.

Sucrose splash test

The Sucrose Splash Test was performed for investigating anhedonia and motivational behavior, as previously described [38]. Prior to the test, animals were allowed a one-hour period for acclimatization. Then, they were placed in a standard plexiglass cage (15 x 30 x 20 cm), under red light illumination, and sprayed twice with a 10% sucrose solution onto the dorsal fur. Mice were not deprived of food or water before the test. The delay before initiation and the total duration of the grooming behavior were recorded over a 5-minute period using video recording, with the fully automated video tracking technology Ethovision XT 16 employed for observation and analysis.

Novel Object Recognition Test

The Novel Object Recognition Test (NORT) represents a validated test for assessing recognition memory in rodents that exploit their natural tendency to explore novel items [39]. The experimental arena was a plexiglass 48 x 48 x 24.5 cm square box. The test protocol consisted of three sessions: habituation, training (S1), and test trial (S2), each lasting 5 minutes. There was a 3 minute-duration and a 1 hour-long interval, respectively between habituation and training, and between S1 and S2 sessions. During the habituation phase, mice were allowed to explore the empty chamber. Subsequently, in the S1 session, they were exposed to two identical objects in the familiar chamber, to assess possible preferences for a specific side of the arena (bias). In the final S2 session, one of the S1 familiar objects (TF) was replaced by a novel object (TN). The analysis encompasses the measurement of both the total exploration time, calculated as the time spent exploring both the TN and the TF during the test phase, and the discrimination index, calculated as DI = (TN - TF) / (TN + TF). The time of direct interaction was evaluated by the time spent sniffing the novel object (TN) or the familiar object (TF). Following each trial, the arena was cleaned with a 10% ethanol solution to mitigate olfactory cues. The movements of the test mice were recorded and tracked using Ethovision XT 16.

Y-maze test

The Y-maze test was utilized to assess spontaneous alternation behavior and spatial working memory in mice [40]. The apparatus was in white acrylic plastic, with three identical arms measuring 8x30x15 cm, set 120° apart. A T-guillotine door was installed at the gateway of the entrance arm, preventing mouse exit. An entry was counted when a subject placed all four limbs into one of the arms. Each trial lasted ten minutes and was recorded and analyzed using Ethovision XT 16. Spatial working memory performance was quantified by calculating the alternation percentage of triads (ABC, BCA, CAB) over the total number of arm entries [41]. Additionally, the total number of arm entries was recorded to evaluate exploratory behavior.

Open Field test

The Open Field Test (OF) was conducted to measure spontaneous locomotor activity in a circular arena (60 cm diameter) during 30 minutes [42]. This test specifically focused on the normalized distance traveled by mice in the arena central area, defined as a zone 5 cm away from the walls. Data acquisition and analysis were performed using Ethovision XT 16. Following the experimental procedure, a comprehensive analysis was conducted on a range of behavioral parameters. This included locomotor activity (total distance traveled), velocity, time spent in the center, number of entries in the center, and number of defecations.

Self-grooming behavior

During spontaneous open-field activity, we additionally examined the self-grooming behavior, scoring the total number of grooming episodes, defined as complete sequences of cleaning and rubbing the face and snout, licking and stroking flanks and abdomen, ano-genitals and tail [43].

Elevated plus-maze test

The Elevated Plus Maze (EPM) test was utilized to evaluate anxiety-like behaviors [44]. The apparatus consisted of a wooden structure comprising two open arms (35 × 6 cm) and two closed arms (35 × 6 × 20 cm) radiating from a central platform. The maze stood 100 cm above the ground. Each mouse was individually placed on the central platform, allowing free exploration for 5 minutes. Video recordings were acquired using a ceiling-mounted camera and analyzed with Ethovision XT 16. The number and the latency of entries into open and closed arms (defined by the entry with all four paws) were scored to measure anxiety levels.

Marble burying test

The marble burying test was utilized to evaluate repetitive and compulsive behaviors [45]. Mice were individually housed in a standard polycarbonate cage measuring 26 x 48 x 20 cm, filled with 5 cm clean bedding. Twenty marbles were tidily laid in four rows on the surface of the bedding. The surface of the bedding was photographed at 5-minute intervals during the 30 minute-long test, to better quantify the number of buried marbles. Marbles were classified as buried if at least three-quarters were covered by the bedding material.

Accelerating Rotarod test

The accelerated rotarod test was used to assess coordination and motor learning. The experimental protocol consisted of three training sessions and one test session, delivered along four consecutive days [46, 47]. The rotarod test was performed using an electronically controlled computerized system (Panlab, Harvard Apparatus), which consists of a rotating cylinder with four lanes (3 cm diameter). Each animal was placed on a rotarod lane. On the first day, mice were acclimated for 30 s to the rod, rotating at 4 rpm constant speed. Then, the rotation velocity was electronically adjusted from 4 to 40 rpm in 300 s with a linear acceleration rate. Each session consisted of 3 trials with 5-minute intervals. The following parameters were measured: latency to fall (seconds); maximal rotational speed reached before falling (rpm); best performance on the last day of testing (maximal latency to fall) [47]. Linear regression analyses were used on the dataset obtained from each mouse, to derive two key parameters: the intercept, as an estimate of initial motor coordination, and the slope, as a proxy of the learning rate [46].

Slice Preparation and Electrophysiology Recordings

Mice (P60-P70) were euthanized by cervical dislocation. Corticostriatal parasagittal and coronal slices (300 µm for intracellular or 220 µm for patch-clamp recordings) were cut with a vibratome in Krebs’ solution, bubbled with 95% O2 and 5% CO2, as previously described [36, 48]. A single slice was transferred into a recording chamber (0.5-1 ml volume), perfused with oxygenated Krebs’ solution at constant temperature (32–33°C) and flow rate (2–3 ml/min). Conventional intracellular recordings were performed blindly with sharp-microelectrodes filled with 2M KCl (40–60 MΩ), as previously described [36, 49]. Signals were acquired using an Axoclamp 2B amplifier and pClamp9 software (Molecular Devices). The recorded neurons were identified as spiny projection neurons (SPNs) according to their electrophysiological properties [36]. Glutamatergic excitatory postsynaptic potentials (EPSPs) were evoked using bipolar electrodes placed in the cortical V–VI layer, in the presence of the GABA_A receptor antagonist picrotoxin (PTX, 50 µM) or bicuculline (10 µM) in the perfusion solution. Stimuli were delivered at 0.05 Hz, with 30 µs duration and intensity evoking 70% maximal amplitude of the EPSP. High-frequency stimulation (HFS), consisting of three trains at suprathreshold intensity (100 Hz frequency, 3 seconds duration, 20 seconds interval), was delivered to induce long-term synaptic plasticity at corticostriatal synapses. Magnesium was omitted from Krebs’ perfusion solution to remove NMDA receptor blockade and optimize induction of long-term synaptic potentiation (LTP) [50]. The EPSP amplitude was continuously recorded for 25 to 40 minutes after HFS and plotted as a percentage of pre-HFS mean control EPSP amplitude. Patch-clamp recordings were performed in the whole-cell configuration, as previously described [48]. Striatal SPNs were visualized using BX51WI microscopes (Olympus), with differential interference contrast (IR-DIC) and standard infrared optics, and identified based on their morphology and electrophysiological properties. Electrophysiological signals were detected using Multiclamp 700B amplifiers coupled to pClamp 10.6 software (Molecular Devices). Voltage- and current-clamp recordings were performed with borosilicate glass pipettes (resistance 5–10 MΩ) filled with an internal solution containing (in mM): 125 K⁺- gluconate, 10 NaCl, 1 CaCl₂, 2 MgCl₂, 0.1 BAPTA, 19 HEPES, 0.3 Na-GTP, 2 Mg-ATP; pH 7.35; 300 mOsm. For mGluI receptor-induced long-term depression (DHPG-LTD) experiments, SPNs were clamped at -50 mV holding potential and recorded in the presence of 50 µM PTX or 10 µM bicuculline [34]. For NMDA current recordings, SPNs were clamped at -70 mV holding potential and recorded in the presence of tetrodotoxin (TTX, 1 µM) plus PTX or bicuculline [51]. In order to calculate the NMDA/AMPA ratio, recording pipettes were filled with (in mM): 120 CsMeSO₃, 15 CsCl, 8 NaCl, 10 TEA-Cl, 10 HEPES, 0.2 EGTA, 2 Mg-ATP, and 0.3 NaGTP; pH adjusted to 7.3 with CsOH; 300 mOsm [52]. Excitatory postsynaptic currents (EPSCs) were initially recorded at -70 mV holding potential, in the presence of PTX or bicuculline. Then, the holding potential was brought to + 40 mV, and NMDA-mediated EPSCs were measured in the presence of PTX or bicuculline plus the AMPA receptor antagonist CNQX (10 µM). Drugs were bath applied by switching the perfusion solution to the drug-containing solution using a three-way tap syringe. Picrotoxin (PTX) was from Sigma-Aldrich (Italy). (S)-3,5-Dihydroxyphenylglycine (3,5-DHPG), 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB), arachidonyl-2'-chloroethylamide (ACEA), 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide (AM251), N-methyl-D-aspartic acid (NMDA), 6-Cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX disodium salt), bicuculline, and tetrodotoxin (TTX) were purchased from Tocris-Cookson (UK).

Western Blot

Western blot of striatal lysates was performed as previously described [48, 53]. Dorsal striata were collected from P60-P90 male and female R451C-NL3 mice and respective age- and sex-matched WT littermates. The samples were homogenated in cold extraction buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.25% Na deoxycholate, 5 mM MgCl₂, 0.1% SDS, 1 mM EDTA, and 1% protease inhibitor cocktail (Sigma-Aldrich). Afterwards, the samples were sonicated, kept in ice for 1 h and centrifuged (13,000 rpm, 15 min, 4°C). The protein content was quantified with the Bradford assay (Bio-Rad). DTT and NuPage LDS sample buffer (Invitrogen, Life Technologies) were added to protein extracts and striatal lysates were denatured (95°C, 5 min), and loaded onto 8–10% SDS-PAGE gels. Gels were blotted onto a polyvinylidene fluoride (PVDF) membrane, that was then incubated overnight at 4°C with the following primary antibodies: rabbit anti-mGlu1 receptor (1.1000, D5H10-12551, Cell Signaling), rabbit anti-mGlu5 receptor (1.5000, ab76316, Abcam), rabbit anti-NMDA receptor subunit 2A (NR2A) (1:1000, 4205, Cell Signaling), rabbit anti-NMDA receptor subunit 2B (NR2B) (1:1000, 4207, Cell Signaling), rabbit anti-AMPA receptor subunit 1 (GluA1) (1:1000, PA5-32425, Invitrogen), rabbit anti-AMPA receptor subunit 2 (GluA2) (1:1000, AB1768-I, Sigma Aldrich), rabbit anti-SHANK3 (1:1000, GTX133163, GeneTex), mouse anti-PSD95 (1:20,000, MAB1598, Millipore), rabbit anti-NL3 (1:8000, GTX131085, GeneTex). Mouse anti-β actin (1:10.000, A5441, Sigma-Aldrich) was incubated 1h at room temperature (RT). Anti-mouse and anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies were used (GE Healthcare). Immunodetection was performed with ECL reagent (GE Healthcare) and iBright CL1000 instrument (Thermo Fisher). Quantification was achieved by ImageJ software (NIH).

Preparation and Western blot analysis of synaptic plasma membranes

Synaptosomes were obtained from the dorsal striata of three-month old control (WT) and R451C-NL3 mice. Frozen brain samples were allowed to thaw on ice, weighed, and homogenized in 10% (w/v) 0.32 M sucrose buffer containing ethylenediaminetetraacetic acid (EDTA 1 mM, Sigma), Tris-HCl (10 mM, pH 7.4, Sigma), phenylmethanesulfonyl fluoride (PMSF 0.5 mM, Sigma) and protease inhibitor cocktail (Roche). The homogenate was centrifuged for 10 min at 1000 x g (4°C) to separate the nuclear pellet. The supernatant was then centrifuged for 30 min at 18,000 x g (4°C), and the resulting pellet was resuspended in 0.32 M sucrose buffer, layered on top of discontinuous sucrose gradient (0.8, 1.0, 1.2 M), and centrifuged for 2 h at 75,000 x g (4°C). The synaptic plasma membrane fractions were stratified at the interface between 1/1.2 M sucrose. The recovered synaptosomes were diluted 1:2 in cold sucrose buffer and centrifuged for 1 h at 40,000 x g (4°C). The pellets were resuspended in Hepes buffer (0.5 mM PMSF, 10 mM Hepes and protease inhibitor cocktail; pH 7.4). A 5 µl volume of the suspension was used to determine the protein content by using the BCA method, and the remaining was stored at -80°C until use. For western blot analysis, 10 µg of proteins from WT and R451C-NL3 samples were denatured in sample buffer (2X) at 37°C for 5 min, separated by 7.5% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (Hybond C-extra, Amersham Biosciences) with a trans-blot semi-dry transfer cell (Bio-Rad). The filters were processed with Western Breeze Chemiluminescent Immunodetection System kit (Invitrogen), as indicated by the manufacturer. In brief, filters were blocked for 30 min with blocking solution and incubated overnight with the following primary antibodies: anti-mGlu5 receptor (rabbit 1:2000, Abcam, cod ab76316), anti-mGlu1α (mouse, 1:1000, G209-488 BD Pharmingen), anti-NL3 (rabbit 1:1000, SYSY 129113, Synaptic Systems), anti-Homer pan (1:1000, Sc-15321, Santa Cruz), and anti-βactin (mouse 1:1000, 4970, Cell Signaling). Alkaline phosphatase-conjugated secondary anti-rabbit or anti-mouse antibodies from Invitrogen kit were used. Chemiluminescence was detected and quantified by VersaDoc™ 4000 Imaging System (Bio-Rad).

Shotgun proteomics analysis of dorsal striatum tissue

The analysis was performed on proteins obtained from the dorsal striatum of WT and KI mice (N = 5 animals/genotype). Proteins (extracted as previously described in the "western blot" section) were transferred to a Microcon-10 centrifugal filter with 10kDa cut off (Millipore) to perform trypsin digestion according to the filter-aided sample preparation (FASP) protocol, as previously described [54, 55]. The filtered buffer was replaced with Urea Buffer (UB: 8M Urea, 100mM Tris-HCl in water, pH 8.5), and the denatured proteins reduced in 8mM DTT in UB (15min at 56°C) and alkylated in 0.05M IAA in UB (20min at room temperature). UB was then replaced by 0.05M ammonium bicarbonate solution (AmBic) in water and proteolytic digestion with trypsin, (with an enzyme/protein ratio of 1:50 (w/w)) at 37°C, for 16–18 hours. Trypsin digestion was blocked by adding formic acid (FA) to a final concentration of 0.2% (v/v), and peptides were recovered from the filter in 0.05M AmBic, concentrated in a speedvac and stored at -80°C until use. Digested peptides were diluted in a 0.1% FA solution in order to load 0.3 µg of each sample, spiked with 500 fmol of EColiClpB Hi3 standard (Waters) on a Symmetry C18 Trap Column, 100Å, 5 µm, 180 µm x 20 mm (Waters). Peptides were thus separated by a 125 min reverse phase gradient at 1.2 µl/min (linear gradient, 2–40% ACN over 90 min) using a HSS T3 100 Å 1.8µm, 150 µm X 100 mm iKey (Waters) maintained at 40°C on an ACQUITY M Class UPLC system (Waters). Separated peptides were analyzed in a shotgun experiment on a Synapt G2-Si Mass spectrometer (Waters) directly coupled to the chromatographic system. Data were acquired in High Definition MS^E (HD MS^E), a data independent acquisition (DIA) protocol where ion mobility separation (IMS) has been integrated into LC- MS^E workflow [54, 56]. Mass spectra were acquired in positive polarity and resolution analyzer mode. TOF MS was operating over 50–2000 m/z using a scan time of 0.5 s and a continuum data format. Data were post-acquisition lock mass corrected using the doubly charged monoisotopic ion of (Glu1)-Fibrinopeptide B (Waters) sampled every 30 s. For IMS, wave height at 40 V, wave velocity of 1000 m/s and transfer wave velocity of 175 m/s were applied. Instrument settings were defined to apply a drift time specific transfer collision energy ramp as previously described [57]. Data from three replicate experiments for each sample were processed for qualitative and quantitative analysis using the Progenesis QI for Proteomics v4.1 software (Waters). The qualitative identification of proteins was obtained by searching in the Uniprot database restricted to mouse taxonomy (Uniprot 2023_04, restricted to Mus Musculus taxonomy), to which the sequence of the EColi ClpB protein (P63285) was appended. Search parameters were set as: 6 ppm for peptide tolerance and 10 ppm for fragment tolerance, minimum one fragment ion matched per peptide, minimum three fragment ions matched per protein, minimum one peptide matched per protein, one missed cleavage accepted, carbamidomethylation of cysteines as fixed modification and oxidation of methionines as variable modification, false discovery rate (FDR) of the identification algorithm at 1%. Label-free quantitative analysis was obtained by using the “Absolute quantification using HiN” option integrated in the software [58, 59] that averaged the most abundant n peptides (n = 3) for each protein to provide a reading for the protein signal, using the Hi3 E. coli standard (Waters) as reference. Protein quantifications were averaged feature-wise per technical replicate to obtain one column per sample in the raw quantification table. Differential expression was performed by fitting a linear model over the log-transformed normalized level of expression. Differentially expressed protein were selected with a threshold for significance set to p-values ≤ 0.05 (≥ 1.3 in -log 10 scale) and |log 2 (MFC)| ≥ 0.3 (equal to 1.3-fold increase/decrease). The mass spectrometry proteomics data will be deposited to the ProteomeXchange Consortium via the PRIDE partner repository [60].

Statistical Analysis

Statistical analysis was performed with Prism software (GraphPad). For the electrophysiological experiments, data analysis was performed offline using Clampfit 10 software (pClamp 10, Molecular Devices). The normality of data distribution was investigated with the Shapiro-Wilk test. For single comparisons of parametric data paired or unpaired t-test was used, whereas non parametric data were analyzed by Mann-Whitney tests. One-way ANOVA followed by Sidak’s multiple comparison test, was utilized to analyze multiple datasets with normal distribution. Two-way ANOVA, followed by Sidak’s multiple comparison test, was used for time-course analysis of the open field, marble and rotarod tests and interaction time comparisons in the 3-chamber test. Statistical significance is set at p < 0.05 (*p < 0.05, **p < 0.01). Data are presented as mean ± standard error of the mean (SEM).

The R451C mutation in the neuroligin 3 gene induces an ASD-like behavioral phenotype in mice.

In addition to social impairments and stereotyped behaviors, the two major phenotypes supporting an ASD diagnosis, autistic children may also face difficulties in movement planning during locomotion, further pointing to the striatum as an anatomical substrate of ASD-associated phenotypes [61]. Previous work implicated striatal abnormalities as the basis of the social deficits and the restricted and repetitive behaviors characteristic of ASD [62–65]. Indeed, the striatum is anatomically and functionally organized in a ventral part, mediating limbic functions of motivation, reward, and emotion related behaviors, and a dorsal region, implicated in cognitive and motor functions, such as sensorimotor processing, goal-directed and automated behavior [66]. Such functional segregation relies on the topographical organization of the glutamatergic inputs to the striatum, which are distributed in a dorsomedial-to-ventrolateral arrangement from the sensorimotor, associative, and limbic cortices [67]. In particular, the dorsal striatum represents a key brain region for the initiation and control of movements, as well as for habitual and repetitive actions [68–76]. Despite this evidence, while there is a substantial amount of work on the role of the ventral striatum in ASD, the dorsal region has so far been poorly investigated.

We therefore analyzed the ASD-like phenotype of R451C-NL3 knock-in (KI) mice using a comprehensive battery of tests focusing on striatal-dependent motor, social and stereotyped behaviors. Accordingly, restricted interests and stereotypies arise from striatal dysfunction [62–65]. We assessed repetitive behaviors of R451C-NL3 mice by evaluating self-grooming during spontaneous activity in the open field test. Normal grooming behavior involves brief bouts lasting seconds to minutes of scratching and fur cleaning using the forelimbs [77]. We observed a significant increase in the number of complete self-grooming sequences in R451C-NL3 mice, with respect to WT littermates, indicating the expression of a stereotypic behavior in mutants (Fig. 1A₁; WT = 5.250 ± 0.729, N = 12; R451C-NL3 = 7.30 ± 0.668, N = 10; Mann-Whitney test, *P_value= 0.022). We then utilized, for the first time in the R451C-NL3 mouse model, the marble-burying, a consolidated test in the study of rodent stereotypic behaviors [2, 45, 78]. Interestingly, this test highlighted a significant increase in the digging and burying behavior of mutants, compared to WT mice (Fig. 1A₂,A₃), further supporting an increase in repetitive behaviors. Specifically, the two-way ANOVA analysis showed a significant main effect of genotype (F(1,27) = 12.60, **P_value= 0.001), with a significant increase in the number of marbles buried within all the evaluated time-intervals (Fig. 1A₂; Sidak's multiple comparisons post-hoc analysis: 0–5 min, **P_value= 0.009; 5–10 min, **P_value= 0.008; 10–15 min, *P_value= 0.043; 15–20 min, *P_value= 0.019; 20–25 min, P_value= 0.186; 25–30 min, *P_value= 0.033). Overall, throughout the test duration, R451C-NL3 mice buried significantly more marbles compared to their WT littermates (Fig. 1A₃; WT = 12.0 ± 1.173, N = 16; R451C-NL3 = 16.23 ± 0.752, N = 13; Mann-Whitney test, **P_value= 0.004). We next investigated social behavior, the second core domain disrupted in ASD, which is based on the ventral striatum [79, 80]. To this aim, in order to evaluate social and novel preference of the mutant mice (Fig. 1B_1 − 4) we employed the 3-chamber test, which is well-established to assess potential alterations in social behavior. R451C-NL3 mice showed no bias for either side of the chamber. When allowed to freely explore, KI mice spent significantly less time than WT littermates investigating the social stimulus and did not exhibit a significant preference for the social stimulus over the non-social stimulus (Fig. 1B₁; Interaction time: WT_object= 60.08 ± 6.811s, WT_social= 104.8 ± 10.81s, N = 10, Sidak’s multiple comparisons test *P_value= 0.018; R451C-NL3_object = 75.20 ± 10.16s; R451C-NL3_social = 80.11 ± 12.42s, N = 9, Sidak’s multiple comparisons test P_value= 0.9997; two-way ANOVA, F(1,34)_interaction = 5.978, P = 0.448). R451C-NL3 mice accordingly displayed a significantly lower social preference index than WT mice (Fig. 1B₂; WT_{social preference index}= 0.633 ± 0.028, R451C-NL3_{social preference index}= 0.509 ± 0.038; unpaired t-test *P_value= 0.018). In the second phase of the test, when allowed to freely interact with either a familiar or an unfamiliar mouse, WT mice spent significantly more time sniffing the unfamiliar mouse, whereas mutant mice showed no difference in the interaction time spent with either the familiar or the novel mouse (Fig. 1B₃; Interaction time: WT_familiar = 60.79 ± 7.317s, WT_novel= 104.5 ± 15.46s, N = 10, Sidak’s multiple comparisons test, *P_value= 0.037; R451C-NL3_familiar = 76.54 ± 8.005s; R451C-NL3_novel = 52.41 ± 10.12s, N = 9, Sidak’s multiple comparisons test, P_value= 0.586; two-way ANOVA, F(1,34)_interaction = 9.670, **P_value= 0.0038). Hence, R451C-NL3 mice showed a reduced novel preference index compared to control animals (Fig. 1B₂; WT_{novel preference index}= 0.615 ± 0.049, R451C-NL3_{novel preference index}= 0.400 ± 0.056; Mann-Whitney test *P_value= 0.017). It is noteworthy that both clinical studies and preclinical research involving model organisms suggest that motivational impairments in ASD extend beyond social rewards and manifest in response to various nonsocial stimuli [81]. To analyze nonsocial motivation, we performed the sucrose splash test. Mice carrying the R451C-NL3 mutation showed a reduced dorsal self-grooming activity, compared to WT littermates (Fig. 1C₁; WT = 10.42 ± 0.857, N = 12; R451C-NL3 = 7.8 ± 0.841, N = 10; unpaired t-test *P_value= 0.043), although no differences were observed in the grooming latency compared to their WT littermates (Fig. 1C₂; WT = 38.75 ± 7.072 s, N = 12; R451C-NL3 = 41.70 ± 6.414 s, N = 10; unpaired t-test P_value= 0.765). Additionally, to exclude cognitive issues as a potential explanation of genotype differences, we assessed the recognition and spatial working memory of the mice using the NORT and Y-maze test, respectively. No significant differences were observed in either task: R451C-NL3 mice were able to discriminate between novel and familiar objects in the NORT test (suppl. Figure 1A₁, A₂), and exhibited normal spontaneous alternations in the Y-maze, indicating preserved spatial working memory (suppl. Figure 1B₁). Analysis of the Y-maze test revealed a significant increase in the total arm entries of R451C-NL3 mice with respect to WT mice, suggesting a hyperactive phenotype of the mutant mice (suppl. Figure 1B₂). Hyperactivity of R451C-NL3 mice was observed also in the open field test, where mutants showed a significant increase in the total horizontal distance traveled (Fig. 1D₁; WT = 119.0 ± 8.376 m, N = 12; R451C-NL3 = 151.2 ± 6.640 m, N = 10; unpaired t-test **P_value= 0.009), and in the horizontal distance traveled in the last two time-intervals of the session (Figs. 1D₂; Sidak’s multiple comparisons test 20–25 min, *P_value= 0.011; 25–30 min, *P_value= 0.043; Two-way ANOVA: main effect of time (F (2.116, 42.31) = 57.53, ****P_value< 0.0001, and genotype (F(1,20) = 8.528, **P_value= 0.0085). The average speed detected during open field test was not significantly different between the two genotypes (Fig. 1D₃; WT = 0.097 ± 0.005 m/s, N = 12; R451C-NL3 = 0.108 ± 0.004 m/s, N = 10; unpaired t-test P_value= 0.112), suggesting that the increased locomotor activity of R451C-NL3 mice might be caused by striatal circuitry impairments affecting the ability to set goals, maintain a direction, and follow a smooth trajectory, rather than to enhanced motor skills [61]. To further explore motor function, we examined coordination and learning with the accelerated rotarod test. The performance improved similarly across the training sessions in mutant and control mice (Fig. 1E₁; two-way ANOVA, F (4.707, 127.1)_time= 37.08, ****P value < 0.0001). However, neither the genotype (Fig. 1E₁; two-way ANOVA F(1, 27)_genotype= 1.166, P_value= 0.289), nor the interaction between genotype and training (Fig. 1E₁; two-way ANOVA F (11, 297)_interaction= 1.190, P_value= 0.293) had a significant impact on the observed improvement. The linear regression analysis did not detect any significant differences between the genotypes in the initial coordination (Fig. 1B₂; WT = 8.667 ± 0.724 r.p.m., N = 22; R451C-NL3 = 8.711 ± 0.784 r.p.m., N = 23; unpaired t-test, P_value= 0.967) or learning rate (Fig. 1B₃; WT = 0.881 ± 0.099, N = 22; R451C-NL3 = 1.009 ± 0.099, N = 23; unpaired t-test, P_value= 0.365). Neverthless, R451C-NL3 mice showed a significantly better performance on the test day (day 4) compared to WT littermates (Fig. 1B₄; WT = 129.0 ± 7.904 s, N = 22; R451C-NL3 = 155.4 ± 10.29 s, N = 19; unpaired t-test, *P_value= 0.045). These findings further point to an impairment of dorsal striatal circuits, which are essential for motor skill acquisition and motor learning [46].

Finally, anxiety-like behaviors were analyzed in both the open field and the EPM, to rule out a possible influence on the results of R451C-NL3 mice in the striatal-dependent behavioral tests (suppl. Figure 1A,B).

Altered protein expression profile in the dorsal striatum of R451C-NL3 mice.

To investigate possible alterations induced by the R451C-NL3 mutation in the dorsal striatum, we first analyzed the protein expression profile, by performing a proteomic analysis. A comparative analysis of differentially expressed proteins (DEPs) in dorsal striatum extracts of control and mutant mice was carried out using LC-MS-based shotgun proteomics. Overall, we quantified 911 proteins in the two mouse groups (N = 5 R451C-NL3; N = 5 WT) and selected 93 DEPs (suppl. Tables S1, S2) with a statistically significant maximum fold change (MFC) of protein expression level set at ≥ 1.3 (ANOVA p value ≤ 0.05). A hierarchical clustering analysis of DEPs was then performed, utilizing Euclidean correlation as the distance metric that is shown in a heat map dendrogram (Fig. 2A). This graphical representation (based on protein recurrence) illustrates the different protein expression in WT vs. KI striatum. Additionally, a volcano scatter plot was employed for rapid visual recognition of proteins exhibiting statistically significant fold change in each comparison (Fig. 2B).

Notably, our analysis showed a marked reduction of NL3 protein abundance (Fig. 2C₁; WT = 14.59 ± 3.113, N = 5; R451C-NL3 = 5.482 ± 0.850, N = 5; unpaired t-test, *P_value= 0.022) within the dorsal striatum of KI, compared to WT, mice. Accordingly, the Western blot analysis showed that the R451C mutation causes NL3 protein downregulation also in the dorsal striatum (Fig. 2C₂; WT = 0.955 ± 0.047, N = 3; R451C-NL3 = 0.217 ± 0.008, N = 3; unpaired t-test, ***P_value= 0.0001), similar to what previously reported in different brain areas of this mouse model [82, 83], as well as in heterologous expression cellular systems (De Jaco et al., 2010). Subsequently, the dataset of identified DEPs was utilized to conduct gene ontology enrichment analysis utilizing ShinyGO 0.80, a gene set enrichment tool (https://bioinformatics.sdstate.edu/go/) [84]. The enrichment analysis of DEPs within the dorsal striatum of R451C-NL3 mice identified several dysregulated pathways with respect to WT animals (suppl. Figure S2A,B). Notably, some of the pathways identified in our proteomic analysis are strongly linked to signal transduction pathways, including G protein-coupled receptor (GPCR)-mediated signaling, neurotransmitter receptors, and postsynaptic signal transduction. These observations are in line with experimental data supporting the pivotal role of NL3 in AMPA and NMDA receptor-mediated excitatory transmission in hippocampus and somatosensory cortex [85–87], in long-term synaptic plasticity [36, 86] and in dendritic spine turnover [88, 89].

The R451C mutation impairs the expression of corticostriatal synaptic plasticity.

Corticostriatal synapses express different forms of bidirectional plasticity, potentiation and depression, that can be induced experimentally by high-frequency stimulation (HFS) of afferent fibers [50], and by agonist-induced mGluI receptors activation [34]. As we previously showed [36], the high-frequency stimulation (HFS) of afferent fibers was not able to induce an activity-dependent LTD at dorsal striatum synapses of R451C-NL3 mice (Fig. 3A; EPSP amplitude post-HFS: WT_POST= 51.43 ± 4.78% of pre-HFS control, n = 18; paired t-test, **** P_value< 0.0001; R451C-NL3_POST = 101.54 ± 3.74% of pre-HFS control, n = 17; paired t-test, P_value= 0.684; WT_POST vs. R451C-NL3_POST, unpaired t-test, ****P_value< 0.0001). We therefore investigated the other forms of long-term synaptic plasticity that can be induced at corticostriatal synapses. We found that activity-dependent long-term potentiation (HFS-LTP), is also impaired in the R451C-NL3 dorsal striatum. Notably, delivery of the induction protocol in R451C-NL3 slices failed to induce an LTP, of amplitude similar to the one observed in WT (Fig. 3B₁,₂; EPSP amplitude post-HFS: WT_POST= 141.4 ± 2.427% of pre-HFS control, n = 6; paired t-test, ****P_value< 0.0001; R451C-NL3_POST = 106.4 ± 0.634% of pre-HFS control, n = 6; paired t-test, ***P_value= 0.0002; WT_POST vs. R451C-NL3_POST; unpaired t-test, ****P_value< 0.0001). Interestingly, several lines of evidence suggest a pathophysiological role of mGlu receptors in ASD [9]. We therefore examined the expression of mGluI-dependent pharmacological LTD in R451C-NL3 mice. Bath application of 50 µM (RS)-3,5-dihydroxyphenylglycine (3,5-DHPG; 10 minutes) resulted in a sustained LTD of the excitatory postsynaptic currents (EPSCs) in WT slices. Conversely, the same protocol in KI slices induced only a transient decrease of EPSC amplitude, which returned to levels not significantly different from baseline (Fig. 3C₁,₂; EPSC amplitude post-DHPG: WT_POST−DHPG = 65.35 ± 4.529% of pre-DHPG control, n = 5, paired t-test, **P_value= 0.0016; R451C-NL3_{POST − DHPG} = 98.31 ± 0.530% of pre-DHPG control, n = 8, paired t-test, P_value= 0.843; WT_POST−DHPG vs. R451C-NL3_{POST − DHPG}, unpaired t-test, *P_value= 0.011).

Exogenous activation of CB1 receptors rescues the expression of mGluI-induced LTD.

We previously showed that the activation of the endocannabinoid (eCB) signaling was able to induce a partial rescue of HFS-LTD in R451C-NL3 slices [36]. We therefore investigated whether the exogenous activation of the eCB system was able to rescue also the pharmacological LTD, induced by 3,5-DHPG application, in KI mice. Corticostriatal slices from mutant and WT mice were perfused with the highly selective CB1 receptor (CB1R) agonist ACEA (20 µM), 10 min before and during DHPG perfusion. The magnitude of DHPG-LTD in control slices was not modified by perfusion with ACEA, suggesting that activation of mGluI receptors induces a production of eCB sufficient for LTD expression in the WT striatum (Fig. 4A₁,₂; EPSC amplitude post-DHPG: WT_POST−DHPG = 66.68 ± 4.958% of pre-DHPG control, n = 7; paired t-test, ***P_value= 0.0005). In KI slices, exogenous CB1R activation was able to fully rescue the expression of DHPG-LTD, which showed a magnitude comparable to the pharmacological LTD of WT slices (Fig. 4A₁,₂; EPSC amplitude post-DHPG: R451C-NL3_{POST − DHPG} = 72.69 ± 7.350% of pre-DHPG control, n = 4; paired t-test, *P_value= 0.033; WT_POST−DHPG vs. R451C-NL3_{POST − DHPG}, unpaired t-test, P_value= 0.500). In line with these observations, DHPG-LTD expression was prevented by slice preincubation with the CB1R-specific antagonist AM-251, and only a mild depression of synaptic transmission was observed in both genotypes (Figs. 4B₁,₂; EPSC amplitude post-DHPG: WT_POST−DHPG = 88.77 ± 7.311% of pre-DHPG control, n = 6, paired t-test, P_value= 0.177; R451C-NL3_{POST − DHPG} = 96.36 ± 2.863% of pre-DHPG control, n = 5; paired t-test, P_value= 0.273; WT_POST−DHPG vs. R451C-NL3_{POST − DHPG}, unpaired t-test, P_value= 0.380).

Overall, these observations show a significant impairment of different forms of long-term synaptic plasticity, encompassing activity-dependent and pharmacological-induced, in the dorsal striatum of R451C-NL3 mice. In particular, the transient depression of EPSC amplitude induced by 3,5-DHPG and the rescue of pharmacological LTD obtained with the CB1R agonist ACEA suggest an impairment of mGluI-mediated signaling in R451C-NL3 mice. Indeed, mGluI receptors are implicated in eCB production [85], and play a pivotal role in modulating synaptic plasticity at excitatory glutamatergic synapses, by promptly regulating the necessary rise in intracellular calcium, needed for activity-dependent eCB retrograde signaling, and the redistribution of AMPA and NMDA receptors [90, 91].

Activation of mGluI receptors does not potentiate NMDA receptor-mediated currents in R451C-NL3 striatal slices.

To further investigate the function of mGluI receptors in the R451C-NL3 striatum, we performed additional electrophysiology experiments. As observed in different brain regions, in the dorsal striatum the NMDA glutamate receptor-mediated inward currents are potentiated by the simultaneous activation of mGlu5 and NMDA receptors. To verify the reproducibility of the NMDA-mediated currents, during whole-cell patch-clamp recordings from SPNs, NMDA (30 µM, 30 s) was applied twice, 5 min apart. NMDA induced transient inward currents of similar amplitude in SPNs from WT and R451C-NL3 mice (data not shown; WT = − 28.9 ± 2.802 pA, n = 17; R451C-NL3 = − 29.86 ± 3.55 pA, n = 24; Mann-Whitney test P_value = 0.923). After incubation with 3,5-DHPG (50 µM, 5 min), a significant enhancement of the NMDA-mediated inward current was observed in WT SPNs, but not in KI slices (Fig. 5A; WT_DHPG= 160.1 ± 13.70% of pre-DHPG amplitude, n = 7, paired t-test **P_value = 0.004; R451C-NL3_DHPG = 103.6 ± 2.868% of pre-DHPG amplitude, n = 9, paired t-test P_value = 0.250). In a further set of experiments, we additionally verified whether an mGlu5 receptor positive allosteric modulator (PAM), CDPPB, was able to potentiate the NMDA receptor-mediated responses in SPNs from mutant mice. As shown in Fig. 5B, similar to 3,5-DHPG, 50 µM CDPPB increased NMDA receptor-mediated inward currents in WT slices, whereas it failed to enhance the NMDA-induced current response in SPNs of R451C-NL3 mice (WT_CDPPB= 162.3 ± 12.68% of pre-CDPPB amplitude, n = 3, paired t-test *P_value = 0.039; R451C-NL3_CDPPB = 109.3 ± 6.26% of pre-CDPPB amplitude, n = 4, paired t-test P_value = 0.236). These data show that the positive modulation exerted by activation of mGlu5 receptors on NMDA-mediated responses is disrupted by the R451C-NL3 mutation. To verify if this alteration was dependent on a dysfunction of NMDA receptors, we further analyzed the ionotropic glutamate-mediated postsynaptic currents in SPNs. Our findings did not show significant differences in the AMPA/NMDA ratio between WT and R451C-NL3 slices (Fig. 5C; WT = 2.739 ± 0.287, n = 5; R451C-NL3 = 2.732 ± 0.273, n = 6; Mann-Whitney test, P_value= 0.931). AMPA- and NMDA-mediated current kinetics were also similar in the two genotypes (Fig. 5D₁,₂; V_H= -70 mV, AMPA rise time: WT = 3.385 ± 0.195 ms, R451C-NL3 = 3.995 ± 0.497 ms, unpaired t-test P_value= 0.297; V_H = + 40 mV, NMDA rise time: WT = 10.13 ± 1.387 ms, R451C-NL3 = 9.987 ± 1.005 ms, unpaired t-test P_value= 0.894; figure E₁,₂;F₁,₂; V_H= -70 mV, AMPA decay time: WT = 27.32 ± 1.043 ms, R451C-NL3 = 38.0 ± 5.369 ms, unpaired t-test P_value= 0.098; AMPA weighted tau: WT = 9.748 ± 0.990 ms, R451C-NL3 = 13.70 ± 2.857 ms, unpaired t-test P_value= 0.329; V_H = + 40 mV, NMDA decay time: WT = 396.3 ± 84.09 ms, R451C-NL3 = 466.5 ± 99.65 ms, unpaired t-test P_value= 0.610; NMDA weighted tau: WT = 166.9 ± 31.49 ms, R451C-NL3 = 233.6 ± 29.79 ms, unpaired t-test P_value= 0.175). Overall, these data indicate that the R451C mutation does not affect either the AMPA or the NMDA receptor function, hence suggesting an impairment of mGlu5 receptor function.

R451C-NL3 mice exhibit a partial postsynaptic remodeling in the dorsal striatum.

Overall, our findings suggest an impairment of mGluI receptor signaling in the dorsal striatum of R451C-NL3 mice, in line with several reports indicating alterations in the function of these receptors, particularly of mGlu-dependent LTD, in different experimental models of ASD [92, 93]. We first examined the protein levels of mGlu5 and mGlu1 receptors in total lysates of the dorsal striatum of WT and R451C-NL3 mice. We observed a slight, nonsignificant decrease in both mGlu1 and mGlu5 receptor subtypes (Fig. 6A,B; mGlu5: WT = 1.000 ± 0.090, N = 8; R451C-NL3 = 0.812 ± 0.069, N = 8; unpaired t-test P value = 0.121; mGlu1: WT = 1.000 ± 0.059, N = 6; R451C-NL3 = 0.853 ± 0.086, N = 6; unpaired t-test P value = 0.191). MGlu5 and mGlu1 receptors are mainly postsynaptic. PSD-95 and SHANK3 play a critical role in organizing and scaffolding signaling molecules at the PSD, influencing receptor trafficking, anchoring, and expression [94, 95]. Notably, we observed a significant increase in PSD-95 protein level in the dorsal striatum of R451C-NL3 mice (Fig. 6C; WT = 1.000 ± 0.045, N = 8; R451C-NL3 = 1.288 ± 0.0823, N = 10; unpaired t-test *P_value= 0.011), whereas the protein level of SHANK3 was significantly reduced (Fig. 6D; WT = 1.000 ± 0.137, N = 7; R451C-NL3 = 0.549 ± 0.073, N = 6; unpaired t-test *P_value= 0.019). Next, we examined the protein levels of subunits within the glutamate receptor family. Specifically, we focused on the NMDA (NR2A and NR2B) and AMPA (GluA1 and GluA2) protein expression levels in the dorsal striatum of WT and R451C-NL3 mice. Our analysis ruled out differences between the two genotypes in the expression levels of either NR2A (Fig. 6E; WT = 1 ± 0.053, N = 3; R451C-NL3 = 1.155 ± 0.225, N = 3; unpaired t-test P_value= 0.541) or NR2B (Fig. 6F; WT = 1 ± 0.084, N = 6; R451C-NL3 = 1.072 ± 0.152, N = 5; unpaired t-test P_value= 0.674). Likewise, our experiments reveal no differences in the expression levels of the AMPA receptor subunits GluA1 (Fig. 6G; WT = 1 ± 0.105, N = 5; R451C-NL3 = 0.853 ± 0.042, N = 6; unpaired t-test P_value= 0.195) and GluA2 (Fig. 6H; WT = 1 ± 0.156, N = 3; R451C-NL3 = 1.021 ± 0.062, N = 3; Mann-Whitney test P_value= 0.700). To determine the regional specificity of the observed protein expression changes, we performed a complementary Western blot analysis in the cortical region. This analysis examined the levels of mGluR5 and PSD-95. Our findings revealed no significant changes in the expression levels of either protein (suppl. figure S3A, 3B). Overall, our findings show that the R451C mutation in the NL3 gene impairs the function of the striatal glutamatergic synaptic machinery.

Altered expression of mGluI-associated synaptic proteins in the R451C-NL3 striatum.

Given the role of perisynaptic mGlu receptors in plasticity [26, 96], we analyzed their specific expression in synaptosomal preparations obtained from the dorsal striatum of both R451C-NL3 and WT mice. Western blot experiments showed a statistically significant decrease of mGlu5 receptor protein levels in R451C-NL3 with respect to WT striatal synaptosomes (Fig. 7A; WT = 100 ± 5.527, N = 6; R451C-NL3 = 66.85 ± 8.036, N = 6; unpaired t-test **P_value= 0.0068). Conversely, synaptic mGlu1 receptor levels were not different between R451C-NL3 and control littermates (Fig. 7B; WT = 100 ± 13,95 N = 4; R451C-NL3 = 80.29 ± 5.504, N = 6; unpaired t-test P_value= 0.1675). Since the synaptic scaffolding protein Homer is a known binding partner of group I mGlu receptors [97, 98] whose expression is required in PSD remodeling, modulation of glutamate receptor-mediated functions, regulation of calcium signaling, and plasticity of excitatory synapses [98, 99], we evaluated whether the reduction in mGlu5 receptor synaptic expression was associated with changes in Homer protein levels. We found no significant difference between R451C-NL3 and WT synaptosomal preparations (Fig. 7C; WT = 100 ± 7.464, N = 6; R451C-NL3 = 83.75 ± 6.764, N = 6; unpaired t-test P_value= 0.1376). In different ASD preclinical models, evidence of mGluI receptor dysfunction has been provided in the cortex [51]. We therefore analyzed the receptor protein levels in cortical synaptosomal preparations from R451C-NL3 and WT mice. Like in the striatum, also in the cortex we observed a significant reduction of mGlu5 receptor level in synaptosomes, whereas mGlu1 receptor and Homer levels were not altered (suppl. figure S3C,D,E; mGlu5: WT = 100 ± 8.254, N = 5; R451C-NL3 = 69.64 ± 5.02, N = 5; unpaired t-test *P_value= 0.0138; mGlu1: WT = 100 ± 6.389, N = 6; R451C-NL3 = 82.89 ± 6.496, N = 6; unpaired t-test **P_value= 0.0895; Homer: WT = 100 ± 6.43 N = 5; R451C-NL3 = 88.11 ± 8.51, N = 5; unpaired t-test P_value= 0.2480). Overall, our findings support a specific impairment of mGlu5 receptor in R451C-NL3 KI mice, suggesting that its dysfunction might underscore the described deficits of striatal synaptic plasticity [100–102].

Neurodevelopmental disorders encompass heterogeneous phenotypes, however genetic studies have shown that these different conditions share a significant enrichment of associate/candidate genes located at the pre- or postsynaptic compartments or known to directly regulate synaptic functions in neurons [103]. These observations are supported by anatomical data obtained from both post-mortem tissue and animal models, showing an altered morphology of dendritic spines and filopodia across different brain regions, leading to the use of the term ‘synaptopathies’ to describe a multitude of conditions, including ASD, directly affecting synaptic processing and plasticity [104]. Despite evidence has been provided that synaptic defects represent a substrate of ASD, we only partially understand the underlying molecular mechanisms [103, 105–107]. Synapse formation is a highly regulated process, involving the gathering of specific cell adhesion molecules and scaffolding proteins, which significantly influence whether an initial contact evolves into an excitatory or an inhibitory synapse [108–110]. Neuroligins play a significant role in defining synaptic properties [111], and their impairment can alter the arrangement of neurotransmitter receptors [112]. In particular, the R451C substitution in the NL3 gene has been implicated in the dysfunction of both inhibitory and excitatory synaptic transmission [107]. Indeed, in the layer 2/3 of the somatosensory cortex of the R451C-NL3 mouse model, an increased frequency of spontaneous inhibitory events, and an impaired endocannabinoid-mediated signaling have been demonstrated [82, 113], whereas, in the same mouse model, an increase of AMPA and NMDA receptor-mediated excitatory synaptic transmission in the CA1 area of the hippocampus [114], and an imbalance in the excitatory/inhibitory postsynaptic activity in pyramidal cells of the basolateral amygdala [115] were reported, together with an impairment of NMDA receptor activity in the medial prefrontal cortex [116]. In the ventral striatum/nucleus accumbens, the R451C-NL3 mutation impairs the synaptic inhibitory transmission onto striatonigral SPNs, but does not affect the expression of DHPG-LTD [46]. Conversely, we previously showed that HFS-LTD expression is impaired at corticostriatal glutamatergic synapses of the dorsal striatum [36]. These observations suggest that the R451C-NL3 mutation produces region-specific effects within the brain, and the striatum in particular. Indeed, this brain area shows a complex dorso-ventral and latero-medial anatomical pattern of cortical inputs, reflecting its involvement in the integration of sensorimotor, cognitive and motivational/emotional information which is the basis for the expression of the motor, sensory, cognitive, and social behaviors affected in ASD [117–120]. Notably, imaging studies have revealed anatomical differences in the striatum of individuals with ASD [121–123]. Accordingly, striatal neurophysiological alterations and neuroanatomical changes have been reported in ASD animal models [107, 124]. Of interest, a dorso-ventral gradient from motor to cognitive, sensory and ultimately goal-directed behaviors has been proposed [125], attributing the generation of stereotypies and repetitive behaviors to the dorsal striatum [68]. Our behavioral analysis of the R451C-NL3 mice, consistent with previous findings, indicates impairments across all the domains relevant to ASD [46, 82, 86, 126]. Notably, in the present study the analysis of R451C-NL3 mice with the marble test disclosed clear stereotypic behaviors, in line with the observed impairment of HFS-LTD in the dorsal striatum [36]. Given the established role of NL3 in neuronal networks, we analyzed the different forms of long-term synaptic plasticity expressed at corticostriatal glutamatergic synapses. As we previously reported, HFS-LTD is impaired in the dorsal striatum of R451C-NL3 mice [36]. Interestingly, also delivery of the LTP induction protocol was not able to induce a significant change in corticostriatal synaptic strength, therefore supporting an impairment of both forms of activity-dependent long-term plasticity. Despite the signaling pathways involved in corticostriatal LTD and LTP expression have not been completely clarified yet, both are believed to involve SPN membrane depolarization, mGluI receptors activation and intracellular calcium increase. Notably, LTD induction is prevented by blockade of mGluI receptors, while it is favored by the activation of these receptors combined to postsynaptic depolarization [127–129]. Similarly, it has been previously shown that mGluI receptors are involved in corticostriatal LTP [100, 130, 131]. Additionally, LTP requires activation of NMDA, muscarinic M1, and either D1 or A2A receptors, insertion of AMPA receptors at the synapse and phosphorylation of DARPP-32 [129], while LTD involves activation of eCB synthesis and release, and activation of presynaptic CB1 receptors, which produces a long-lasting decrease in glutamate release probability [129]. Similarly to HFS-LTD, striatal mGluI-LTD depends on an eCB-mediated inhibition of glutamate release [11], internalization of ionotropic glutamate receptors and modifications of AMPA receptor subunit composition [34, 132]. As further evidence of synaptic plasticity impairment in the dorsal striatum, we therefore additionally investigated mGluI-induced pharmacological LTD. The present work shows that pharmacological, in addition to activity-dependent, synaptic plasticity is also altered in the dorsal striatum of R451C-NL3 mice, further pointing to an involvement of mGluI receptors in the inability of corticostriatal synapses to undergo long-lasting changes. Indeed, our observation that 3,5-DHPG does not potentiate NMDA currents in ASD mice, despite normal expression of ionotropic receptor subunits, suggests that the impairment of LTP may depend on mGluI receptor dysfunction, as observed in other brain regions [133]. Disruption of DHPG-LTD maintenance is also in line with this view. Notably, we show that 3,5-DHPG application induces a transient depression of excitatory synaptic transmission, suggesting dysfunction of the signaling pathway activated by mGluI receptors, which is necessary for DHPG-LTD maintenance. Indeed, we show that exogenous activation of CB1 receptors successfully restores the expression of DHPG-LTD at R451C-NL3 corticostriatal synapses. On the other hand, CB1 receptor inhibition produces only a partial disruption of LTD induced by mGluI activation at WT synapses. These findings suggest that dysfunction of mGluI receptors in the dorsal striatum of R451C-NL3 mice may prevent retrograde eCB signaling, which is essential for LTD expression [134]. These observations are relevant, since a growing amount of work supports the involvement of mGluI, particularly mGlu5, receptors in ASD. The expression of mGluI receptors was upregulated at forebrain synapses of infant rats exposed to valproic acid [4]. MGlu5 receptor-mediated PI hydrolysis was blunted in the cerebral cortex, hippocampus, and corpus striatum of Ube3am-/p + and Fmr1 knockout mice, modeling Angelman and Fragile X syndromes, respectively [7]. Dysregulation of mGlu5 receptor signaling has been reported in the hippocampus of Fmr1 knockout mice [7, 92, 135], whereas tuberous sclerosis mouse models exhibited an impaired mGluI-dependent LTD [136]. MGluI receptors are mainly found at the postsynaptic area, where they functionally and physically interact with ionotropic NMDA receptors [98, 137–139]. Previous studies have provided evidence for a facilitatory role of mGluI receptors on ionotropic NMDA receptor function in striatal neurons [19, 140]. Interestingly, we report that activation of mGlu5 receptors, with either the agonist 3,5-DHPG or the PAM CDPPB, failed to potentiate the NMDA receptor-mediated currents. Remarkably, our research did not uncover variations either in the inward current triggered by NMDA receptor activation, or in the AMPA/NMDA ratio. Furthermore, we did not observe differences in the kinetics of AMPA and NMDA receptor-mediated currents, as well as in the protein expression levels of the respective subunits, further pointing to metabotropic, rather than ionotropic, glutamate receptor dysfunction. Our biochemical (both proteomics and WB) data in the R451C-NL3 mouse model, characterized by a dramatic reduction of NL3 expression [82], confirm this protein reduction also in striatum and demonstrate a significant decrease of the mGlu5 receptor protein level in striatal and cortical synaptosomal preparations, whereas no differences were found in mGlu1 receptor protein levels. Notably, several ASD-associated genes encode mGlu5 receptor-interacting proteins, including SHANK3 and Homer scaffold proteins [141–143]. While mGlu5 receptors potentiate NMDA receptor function, Homer links the mGlu5 receptor to postsynaptic signaling pathways [144], and ensures the proper localization of the NMDA/mGlu5 receptor complex at the cell surface, a process facilitated by neuronal adhesion molecules, such as NL3, NL4 and Neurexin 1 [13, 145], (Fig. 8).

SHANK3 is a scaffolding protein that can interact with both NLs and Neurexins to coordinate signaling between pre- and postsynaptic regions in excitatory glutamatergic synapses [141]. Specifically, it interacts directly with the GKAP protein, which in turn is associated with the NMDAR-PSD95 complex, and, on the other hand, with Homer proteins by forming multimers through the interaction of their coiled-coil domain and linking Shank to mGluRI and inositol triphosphate (IP3) [146]. In addition, Shank proteins bind Homer and PSD-95 complexes in postsynaptic density, regulating trafficking and signaling efficiency of mGluRIs [144, 147, 148]. PSD-95 is a major regulator of synaptic maturation, which stabilizes NMDA and AMPA receptors on the postsynaptic membrane [149, 150]. Notably, we found alterations in the expression of ASD-relevant postsynaptic proteins: a decrease in SHANK3 and an increase in PSD-95 protein levels. Overwhelming evidence correlates PSD-95 disruption to cognitive and learning deficits in ASD [151]. In animal studies, PSD-95 deficiency was associated with alterations in the composition and function of NMDA and AMPA receptors, while increased expression led to redistribution of NL2 from inhibitory to excitatory synapses in neuronal cell cultures [152, 153].

LIMITATIONS

The present study focused on the electrophysiological and molecular properties of dorsal striatum synapses, as bases of key behavioral alterations observed in the R451C-NL3 mouse model of ASD. The NL3 gene is located on the X chromosome. The study was conducted on male hemizygous mice, since ASD cases present a 4:1 prevalence in males vs. females. It would be of interest to analyze females in future studies. However, studies in heterozygous females are complicated by the random X chromosome inactivation leading to mosaicism.

Whereas the proteomic approach allows the detection of hundreds of proteins in a single experiment with high accuracy and resolution, the digested and fragmented peptides are generally mapped in relation to the abundance of each protein in the sample, which means that the most abundant proteins are more likely to be identified, while less abundant proteins might be missed [154].

In conclusion, the present work reports an overall disruption of the glutamatergic postsynaptic density in the dorsal striatum of the R451C-NL3 mouse model of ASD, resulting in the impairment of bidirectional long-term synaptic plasticity. Our data point to a blunted mGlu5 receptor signaling, causing reduced eCB production and impaired NMDA receptor-mediated current enhancement, as culprit of synaptic plasticity dysfunction. Therefore, our work adds to previous evidence on different experimental models of ASD, implicating disrupted mGlu5 receptor signaling in the pathophysiology of ASD, albeit with divergent outcomes in the various brain areas and/or experimental models investigated [107]. Accordingly, mGlu5 receptors have been investigated as possible therapeutic targets in ASD. In accordance with our data, studies reported a rescuing effect of mGlu5 receptor agonists on synaptic plasticity. In particular, SHANK3 knockout mice, similar to our data, showed a mislocalization of mGlu5 receptors at the striatal postsynaptic density, causing an impairment of LTD that was rescued by CDPPB [155]. Surprisingly, the positive allosteric modulator did not ameliorate the behavioral alterations observed in this ASD mouse model, rather the mGlu5 receptor antagonist MPEP reversed the hypoactivity in the open field and the increased self-grooming of SHANK3 mice [155]. This observation, together with other reports describing beneficial effects of mGlu5 receptor antagonists on the behavioral alterations observed in the BTBR, valproic acid and Fmr1 knockout mouse models of ASD [156–158], suggests that the systemic modulation of mGlu5 receptor activity may result in outcomes different from its striatal-specific activation.

3,5-DHPG, (S)-3,5-Dihydroxyphenylglycine

ACEA, arachidonyl-2'-chloroethylamide

AM251,(1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide

AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

ANOVA, Analysis of variance

ASD, autism spectrum disorder

CDPPB, 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide

CNQX disodium salt, 6-Cyano-7-nitroquinoxaline-2,3-dione disodium

D2R, dopamine type 2 receptor

eCBs, endocannabinoids

EPM, Elevated Plus Maze

EPSC, Excitatory postsynaptic current

EPSP, excitatory postsynaptic potential

FXS, Fragile X syndrome

HFS, High-frequency stimulation

ID, intellectual disability

LTD, long-term depression

LTP, long-term potentiation

mGluI, group I metabotropic glutamate

mTOR, mammalian target of rapamycin

NL3, neuroligin 3

NMDA, N-methyl-D-aspartic acid

NORT, Novel Object Recognition Test

OCD, obsessive-compulsive disorder

OF, Open Field Test

PAM, positive allosteric modulator

PKC, protein kinase C

PMSF, phenylmethanesulfonyl fluoride

PSD, postsynaptic density

PTX, picrotoxin

PVDF, polyvinylidene fluoride

SPN, spiny projection neurons

TTX, tetrodotoxin

Ethics approval and consent to participate

Not Applicable

Consent for publication

Not Applicable

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

The work was partially supported by a 2022 Autism Research Institute grant to P.B..

Authors' contributions

MMe, MMo, SDA, LP, MVC and PB were responsible for the conception and design of the experiments. MMe, MMo, SDA, GM, IEA, GP, AT, and LP were responsible for the collection, analysis and interpretation of data. IR, DC, and GS supervised the behavioral analysis and the mouse model management. MMe and PB wrote the manuscript, SDA and MVC critically revised it, and all the authors had the opportunity to contribute to its editing. All the authors are listed and qualify for authorship. All authors read and approved the final manuscript.

Acknowledgements

We wish to thank Mr. Gianni Cantini, Mr. Massimiliano Di Virgilio, Mr. Alessio Fieramosca and Dr. Annarita Wirz for support and assistance with animal care, Mr. Massimo Tolu and Mr. Graziano Bonelli for technical assistance.

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No competing interests reported.

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Impaired metabotropic glutamate type 5 receptor signaling in the dorsal striatum of the R451C-neuroligin 3 mouse model of Autism Spectrum Disorder

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Abstract

Figures

BACKGROUND

METHODS

Experimental Model

Behavioral Assays

Three-chamber social interaction test

Sucrose splash test

Novel Object Recognition Test

Y-maze test

Open Field test

Self-grooming behavior

Elevated plus-maze test

Marble burying test

Accelerating Rotarod test

Slice Preparation and Electrophysiology Recordings

Western Blot

Preparation and Western blot analysis of synaptic plasma membranes

Shotgun proteomics analysis of dorsal striatum tissue

Statistical Analysis

RESULTS

DISCUSSION

LIMITATIONS

CONCLUSIONS

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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