Abstract: Abstract Pathogenic variants in GRIN2D, encoding one of the subunits of the NMDA receptor (NMDAR), are associated with developmental and epileptic encephalopathies (DEEs). Unusual for de novo mutations, the recurrent, de-novo, gain of function, missense mutation c.1999G>A (p.Val667Ile) was discovered in multiple patients. We characterized a mouse model carrying the orthologous Grin2d mutation, using behavioral paradigms, electrophysiological recordings in acute brain slices focusing mainly on the activity of Purkinje neurons (PNs) in the cerebellum, and electrocorticography (ECoG) recordings monitoring brain activity and the response to several drugs. Grin2d mutant mice exhibit a range of phenotypes that closely mirror the human disease, including premature mortality, spontaneous seizures, and early onset of motor deficits followed by cognitive impairment. In addition, we observed complex developmental changes in PNs with reduced spontaneous firing in immature mice and augmented synaptic response to NMDA application in older mice. ECoG recordings demonstrated profound and continuous abnormal brain activity, with altered spectral properties and a prominent narrowband activity in the theta, alpha, and beta bands, paralleling the patterns seen in a patient with the same GRIN2D pathogenic variant. Acute administration of ketamine at a low dose (0.5 mg/kg) had a limited effect on the spectral properties, and higher dosages (4 or 10 mg/kg) caused seizures. Conversely, memantine (10 mg/kg) and phenytoin (30 mg/kg) demonstrated a small corrective effect on ECoG properties. Together, Grin2d mutant mice recapitulate key phenotypes of patients with pathogenic GRIN2D variants, including unique, abnormal brain oscillations, which may serve as a biomarker for quantifying drug responses and guiding future research efforts.

Abstract: Abstract Anti-NMDA receptor (NMDAR) encephalitis is a devastating disease with severe psychiatric and neurological symptoms believed to be caused by pathogenic autoantibodies that bind to the N-terminal domain (NTD) of the NMDAR GluN1 subunit (GluN1-NTD) crosslinking adjacent NMDARs and driving their internalization. Here we describe ART5803, a humanized monovalent antibody, as a potential therapy for anti-NMDAR encephalitis. ART5803 binds with a high affinity (K D = 0.69 nM) to GluN1-NTD without affecting NMDAR activity or inducing internalization. ART5803 blocks NMDAR internalization induced by patients’ pathogenic autoantibodies, and restores NMDAR function. A marmoset animal model was developed using sustained intracerebroventricular (ICV) administration of a human pathogenic autoantibody to evoke behavioral and motor abnormalities. ART5803 ICV infusion or peripheral injections rapidly reversed these abnormalities. These data, together with the pharmacokinetic profile in cynomolgus monkeys, indicate a therapeutic potential for intravenous (IV)-administered ART5803 as a fast-acting and efficacious option for anti-NMDAR encephalitis.

Abstract: Abstract Background Brain-derived neurotrophic factor (BDNF) is a key mediator of synaptic plasticity and memory formation in the hippocampus. However, the BDNF-induced alterations in the glutamate receptors coupled to the plasticity of glutamatergic synapses in the hippocampus have not been elucidated. In this work we investigated the putative role of GluN2B-containing NMDA receptors in the plasticity of glutamatergic synapses induced by BDNF. Methods The effects of BDNF on the surface expression of GluN2B-containing NMDA receptors was investigated in cultured hippocampal neurons and in hippocampal synaptoneurosomes by immunocytochemistry under non-permeabilizing conditions, using an antibody that binds to an extracellular epitope. Long term potentiation of hippocampal CA1 synapses was induced by using θ-burst stimulation. Epileptic seizures were induced using the Li + -pilocarpine model of temporal lobe epilepsy. Pyk2 phosphorylation was assessed by western blot with a phosphospecific antibody. Results Stimulation of hippocampal synaptoneurosomes with BDNF led to a significant time-dependent increase in the synaptic surface expression of GluN2B-containing NMDA receptors as determined by immunocytochemistry with colocalization with pre- (vesicular glutamate transporter) and post-synaptic markers (PSD-95). Similarly, BDNF induced the synaptic accumulation of GluN2B-containing NMDA receptors at the synapse in cultured hippocampal neurons, by a mechanism sensitive to the PKC inhibitor GӦ6983. The effects of PKC may be mediated by phosphorylation of Pyk2, as suggested by western blot experiments analyzing the phosphorylation of the kinase on Tyrosine 402. GluN2B-containing NMDA receptors mediated the effects of BDNF in the facilitation of the early phase of long-term potentiation (LTP) of hippocampal CA1 synapses induced by θ-burst stimulation, since the effect of the neurotrophin was abrogated in the presence of the GluN2B inhibitor Co 101244. In the absence of BDNF, the GluN2B inhibitor did not affect LTP. Surface accumulation of GluN2B-containing NMDA receptors was also observed in hippocampal synaptoneurosomes isolated from rats subjected to the pilocarpine model of temporal lobe epilepsy, after reaching Status Epilepticus, an effect that was inhibited by administration of the TrkB receptor inhibitor ANA-12. Conclusion Together, these results show that the synaptic accumulation of GluN2B-containing NMDA receptors mediate the effects of BDNF in the plasticity of glutamatergic synapses in the hippocampus.

Abstract: The dysconnectivity hypothesis of schizophrenia suggests that atypical neural communication underlies the disorder's diverse symptoms. Building on this framework, we propose that specific synaptic disturbances within thalamo-cortical circuits contribute to an imbalance in excitation and inhibition, leading to alteration in oscillations. Our study investigates these alterations and explores whether synaptic restoration can remediate neural activity of schizophrenia and align it with healthy patterns. We analyzed magnetoencephalography data from schizophrenia patients and healthy controls using dynamic causal modeling to identify synaptic differences in thalamo-cortical circuits. The analysis focused on N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), gamma-aminobutyric acid type A (GABA-A), and gamma-aminobutyric acid type B (GABA-B) receptor-mediated connections. In silico synaptic restoration analysis simulated the effects of targeted adjustments to these receptor-mediated connections to assess whether altered neural activity in schizophrenia could be restored to match control patterns. Schizophrenia patients showed statistically significant differences in increased NMDA receptor excitation in superficial pyramidal neurons and reduced GABA-B receptor inhibition between interneurons and pyramidal cells. Parameter recovery analysis revealed limitations for these specific parameters, suggesting that receptor-level interpretations should be made with caution. The in silico synaptic restoration analysis indicated that coordinated modifications across multiple synaptic pathways could potentially remediate neural activity to resemble healthy controls. This restoration approach suggests the complex nature of synaptic dysfunction in schizophrenia may involve coordinated changes across multiple synaptic parameters rather than isolated alterations. While our findings provide preliminary evidence extending the dysconnectivity theory of schizophrenia, the parameter recovery limitations suggest that specific receptor claims should be interpreted with caution.

Abstract: N-Methyl-d-aspartate (NMDA) receptors, a subtype of ionotropic glutamate receptors in the central nervous system (CNS), have garnered attention for their role in brain disorders. Specifically, GluN2A-containing NMDA receptors have emerged as a potential therapeutic target for the treatment of depressive disorders and epilepsy. However, the development of GluN2A-containing NMDA receptor-selective antagonists, represented by N-(4-(2-benzoylhydrazine-1-carbonyl)benzyl)-3-chloro-4-fluorobenzenesulfonamide (TCN-201) and its derivatives, faces a significant challenge due to their limited ability to penetrate the blood-brain barrier (BBB), hampering their in vivo characterization and further advancement. In this study, we reported a series of 2-((5-(phemylamino)-1,3,4-thiadiazol-2-yl)thio)-N-(cyclohexylmethyl)acetamide derivatives, achieved through a structure-guided optimization strategy using free energy perturbation (FEP) and BBB permeability estimation. Through systematic exploration of various phenyl substitutions, compound 1f emerged as a standout compound, demonstrating substantially enhanced inhibitory activity compared with the lead compound TCN-213. Compound 1f not only displayed satisfactory BBB permeability but also showed antidepressant-like potency in the hydrocortisone-induced zebrafish depression-like model. All results position it as a promising candidate for developing innovative therapeutics for NMDA receptor-related disorders.

Abstract: The NMDA receptor has long attracted researchers' attention due to its potential as a drug target and its central role in the central nervous system. The NMDA receptor is a ligand-gated and voltage-dependent ion channel widely distributed in the central nervous system. In this study, we employed a drug design strategy combining "molecular assembly" and "combinatorial chemistry." By reducing the carbonyl group of germacrone to a hydroxyl group and esterifying it with alanine and linarinic acid, we successfully obtained nine novel germacrone derivatives through two rounds of structural optimization. We evaluated the neuroprotective activity of these nine derivatives using the MTT assay. The results revealed that compound C1 exhibited particularly outstanding activity, achieving a cell protection rate of 29.63 ± 1.56 % at a concentration of 0.05 μM, outperforming the positive control drug, ifenprodil. Further experiments on NMDA-induced Ca2+ influx verified that the action site of compound C1 was the NMDA receptor, and demonstrated its superior antagonistic effect on the NMDA receptor compared to germacrone and ifenprodil. Additionally, molecular docking studies and ADMET property predictions were conducted for compound C1. The results showed that compound C1 tightly bound to the active site of the NMDA receptor and possessed favorable pharmacokinetic properties. In conclusion, compound C1, characterized by a germacrone-linarinic acid structure, not only exhibits strong antagonistic effects on the NMDA receptor but also demonstrates excellent pharmacokinetic properties, indicating its potential for further development as a therapeutic drug for central nervous system diseases.

Abstract: Abstract While N-methyl-d-aspartate receptor (NMDAR) blockade is crucial for the rapid antidepressant effects of ketamine, the involvement of other mechanisms remains contentious, particularly regarding the role of serotonin, a key neurotransmitter in the target of traditional antidepressants. Here, we demonstrate that ketamine elevates serotonin levels by inhibiting the serotonin transporter (SERT). A cryogenic electron microscopy structure of ketamine-bound SERT in the outward-open conformation, resolved at 3.2 Å, indicates that ketamine binds to the central site of SERT. Elevated serotonin, along with NMDAR inhibition, induces ketamine-like rapid antidepressant effects. This increase in serotonin leads to the activation of vasoactive intestinal peptide (VIP)-expressing interneurons, which are essential for the rapid antidepressant effects of ketamine. Inhibition of VIP neurons blocks these effects and ketamine-like effects, highlighting a crucial cell type-specific mechanism. These findings identify a critical pathway in the rapid antidepressant actions of ketamine and offer potential pharmacological strategies for developing rapidly acting antidepressants.

Abstract: Anti-N-methyl-D-aspartate (NMDA) receptor encephalitis is a rare but serious autoimmune disorder. It is often associated with ovarian teratomas and mimics psychiatric or infectious conditions, leading to frequent misdiagnosis. Early recognition and treatment are crucial for better outcomes. We present the case of a 31-year-old woman who developed sudden behavioural changes, seizures, and cognitive decline. Initial tests showed no clear cause. The cerebrospinal fluid analysis confirmed anti-NMDA receptor antibodies, and imaging revealed an ovarian teratoma. Immunotherapy alone failed, and tumour removal was necessary for recovery. Her condition improved significantly after surgery. Our case highlights the difficulty of diagnosing autoimmune encephalitis in young women with unexplained neuropsychiatric symptoms. Recognising paraneoplastic causes and using a multidisciplinary approach are essential for timely intervention. Immunotherapy can help, but it may not be enough without tumour removal. Autoimmune encephalitis requires prompt diagnosis and aggressive treatment. Failure to identify and treat underlying causes can lead to severe complications or delayed recovery. This case emphasises the importance of considering anti-NMDA receptor encephalitis in young women with new-onset neuropsychiatric symptoms. Early cerebrospinal fluid analysis and imaging can confirm the diagnosis and guide treatment decisions.

Abstract: Excitatory glycine receptors (eGlyRs), composed of the glycine-binding NMDA receptor subunits GluN1 and GluN3A, have recently emerged as a novel neuronal signaling modality that challenges the traditional view of glycine as an inhibitory neurotransmitter. Unlike conventional GluN1/GluN2 NMDARs, the distribution and role of eGlyRs remain poorly understood. Here, we show that eGlyRs are highly enriched in the ventral hippocampus (VH) and confer distinct properties on this brain region. eGlyRs display a massive expression in both VH CA1 pyramidal cells and SST- and PV-positive interneurons, whereas in the dorsal hippocampus (DH) pyramidal cells lack these receptors. eGlyRs mediate excitatory tonic currents and control VH network excitability. They are also responsible for the attenuated long-term potentiation (LTP) in the VH compared with the DH, providing a molecular basis for this difference. Furthermore, eGlyRs are required for regulation of VH LTP by corticosterone, pointing to eGlyRs as mediators of the neuroendocrine stress response. Consistent with this pervasive influence in the ventral division of the hippocampus, eGlyRs contribute to the modulation of anxiety-related behaviors. Our work identifies eGlyRs as key players in VH circuitry and function, demonstrating their intimate association with brain regions that control internal states and emotional processing.

Abstract: GluN2B-containing NMDARs are related to neurodegenerative diseases, making the GluN2B-selective antagonist a promising drug candidate.

Abstract: Abstract Aims Diabetic neuropathic pain (DNP) is a debilitating complication of diabetes mellitus that significantly impairs patients' quality of life. In this study, we aimed to investigate whether much higher plasma advanced glycation end products (AGEs) concentrations discriminate between diabetes‐affected individuals with pain and those without pain. Methods We administered exogenous AGEs intravenously to C57BL/6J wild‐type mice and assessed nociceptive behaviours, as well as anxiety‐ and depression‐like behaviours. To explore the further mechanism, we knocked out the protein tyrosine phosphatase 1B (PTP1B) in the spinal dorsal horn by injecting the AAV viral construct encoding short‐hairpin RNA (shRNA). Electrophysiological recordings were used to assay the N‐methyl‐D‐aspartate receptor (NMDAR) function. Results Our results demonstrated that elevated plasma AGEs levels led to significant hyperalgesia, which was alleviated by the specific knockout of PTP1B in the spinal dorsal horn. However, this knockout did not ameliorate AGEs‐induced anxiety‐ and depression‐like behaviours. Electrophysiological recordings revealed that AGEs enhanced NMDAR‐mediated excitatory postsynaptic currents (eEPSCs) in spinal cord slices, an effect attenuated by PTP1B inhibition. Biochemical analyses showed that AGEs decreased phosphorylation at the Tyr529 site of Src kinase and increased phosphorylation at the Tyr1472 site of the NMDAR subunit 2B (GluN2B); these changes were reversed by PTP1B knockdown. Conclusion These findings suggest that elevated plasma AGEs contribute to hyperalgesia through a PTP1B‐dependent mechanism involving Src/NMDAR signalling in the spinal dorsal horn. Targeting the AGEs‐PTP1B‐NMDAR pathway may offer new therapeutic strategies for managing DNP.

Abstract: Recent investigations into the rapid antidepressant effects of ketamine, along with studies on schizophrenia-related susceptibility genes, have highlighted the GluN2A subunit as a critical regulator of both emotion and cognition. However, the specific impacts of acute pharmacological inhibition of GluN2A-containing NMDA receptors on brain microcircuits and the subsequent behavioral consequences remain poorly understood. In this study, we first examined the effects of MPX-004, a selective GluN2A NMDA receptor inhibitor, on behavior within the dorsomedial prefrontal cortex (dmPFC). Local administration of MPX-004 in the dmPFC led to a reduced immobility duration in the forced swim test, an acute antidepressant-like effect, impairments in sensorimotor gating, and a schizophrenia-like phenotype. In vivo multiple-channel recordings and c-Fos staining revealed that MPX-004 decreases the activity of parvalbumin-expressing interneurons (PV-INs) and increases the activity of pyramidal neurons (PYNs). In vivo patch-clamp recordings further confirmed that PV-IN inactivation leads to an elevated PYN firing rate in the PFC. In vitro whole-cell recordings demonstrated that PV-INs receive stronger excitatory synaptic input and respond more robustly to presynaptic stimulation than do somatostatin-expressing interneurons (SST-INs) and PYNs, rendering them susceptible to GluN2A inhibition. Finally, the specific knockdown of GluN2A in prefrontal PV-INs abolished the behavioral effects of MPX-004, underscoring a critical role of the GluN2A-mediated modulation of PV-INs in these phenotypes. Together, these findings reveal that PV-INs are particularly vulnerable to GluN2A inhibition, leading to disinhibition of prefrontal circuits and resulting in both antidepressant-like and schizophrenia-like behaviors.

Abstract: Neocortical circuits use synaptic and intrinsic forms of homeostatic plasticity to stabilize key features of network activity, but whether these different homeostatic mechanisms act redundantly or can be independently recruited to stabilize different network features is unknown. Here, we used pharmacological and genetic perturbations both in vitro and in vivo to determine whether synaptic scaling and intrinsic homeostatic plasticity (IHP) are arranged and recruited in a hierarchical or modular manner within layer 2/3 (L2/3) pyramidal neurons in the rodent primary visual cortex (V1). Surprisingly, although the expression of synaptic scaling and IHP was dependent on overlapping signaling pathways, they could be independently recruited by manipulating spiking activity or NMDA receptor (NMDAR) signaling, respectively. Further, we found that changes in visual experience that affect NMDAR activation but not mean firing selectively trigger IHP, without recruiting synaptic scaling. These findings support a modular model in which synaptic and IHP respond to and stabilize distinct aspects of network activity.

Abstract: While N-methyl-d-aspartate receptor (NMDAR) hypofunction has been suggested as a hallmark of schizophrenia, the role of subunit-specific dysregulation such as GluN2A overexpression remains poorly understood. The present study comprehensively investigated the impact of GluN2A overexpression on behavioral phenotypes, cognitive functions, and synaptic plasticity in transgenic mice with forebrain-specific overexpression of the GluN2A subunit (GluN2A-TG). Behavioral assessments revealed schizophrenia-like phenotypes, including prolonged stereotypic movement duration, impaired sensorimotor gating, reduced social interaction, and diminished nest-building activity in GluN2A-TG mice. Consistently, GluN2A-TG mice exhibited not only deficits in spatial working memory and olfactory working memory but also impaired associative learning. In addition, both long-term potentiation and long-term depression were significantly attenuated in the prefrontal cortex (PFC) of GluN2A-TG mice. Furthermore, electrophysiological analysis of NMDAR-mediated excitatory postsynaptic currents in PFC neurons revealed altered kinetics characterized by a faster decay time and significantly increased amplitude in GluN2A-TG mice. Collectively, these findings suggest that GluN2A overexpression may induce schizophrenia-like phenotypes via impairing NMDAR-dependent long-term synaptic plasticity in the PFC, likely due to altered NMDAR subunit composition leading to disrupted calcium signaling dynamics. These results provide critical insights into the pathological role of GluN2A in schizophrenia.

Abstract: Mutations in N-methyl-D-aspartate receptors (NMDARs) cause epilepsy and profound cognitive impairment, though the underlying subunit-specific vulnerabilities remain unclear. We investigate the impact of a severe human variant in the lurcher motif of obligate GluN1 NMDAR subunit using transgenic mice, revealing unexpected context-dependent phenotypes. We show that the GluN1 Y647S variant significantly reduces current flow through pharmacologically isolated synaptic NMDARs in prefrontal neurons. Yet in intact local circuits, this loss-of-function paradoxically extends NMDAR-dependent dendritic integration, causing prolonged circuit-wide excitation that promotes seizures. Mutant receptors appear deficient in engaging opposing dendritic ion channels that normally curtail NMDAR-dependent excitation. Boosting SK channel activity normalizes dendritic integration, whereas slight decreases in extracellular magnesium further extend abnormally prolonged integration in mutant mice. We find that magnesium supplementation successfully treats seizures in vivo in the transgenic mice, despite loss-of-function of NMDARs. Overall, we disentangle a GluN1 variant's receptor-level effects and its dendritic impact to treat seizures effectively.

Abstract: Anxiety has been described in the initial stages of schizophrenia, and affective flattening in the chronic illness. The etiology remains unknown. Ketamine, a noncompetitive N-Methyl-D-amino-aspartate acid (NMDA) receptor antagonist, is used in rats as a translational model of schizophrenia. A glutamate deficit within Nucleus Accumbens Septi (NAS) afferent projections has been proposed to be involved in schizophrenia. The amygdala is related to memory, fear and anxiety, and is closely linked to the NAS. Here, we studied anxiety in male rats using the elevated plus-maze (EPM) after receiving acute administration of different subanesthetic doses of ketamine. The metabolic state of the amygdala was measured after ketamine treatment. The main aim of the present study is to compare the effect of different doses as emulating progressive stages of schizophrenia. Classical anxiety parameters were observed during elevated plus maze (EPM) experiments in rats with the low doses of ketamine. After this, amygdalas were randomly extracted and submitted to a test of redox cellular metabolic activity with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Low doses (1.25 and 2.5 mg/kg) significantly decreased time spent in the open arm, time per entry and open arm entries, and increased time in the closed arms and grooming. These doses also decreased metabolic activity. We conclude that the administration of subanesthetic doses of ketamine exert an acute anxiogenic effect in the plus maze test at the lower doses, accompanied by a decrease in amygdala metabolic activity, suggesting metabolic exhaustion. The higher doses reversed the anxiety parameters, suggesting an explanation of the opposite symptoms in schizophrenia progression.

Abstract: Patients with Duchenne muscular dystrophy (DMD) may experience neurobehavioral and cognitive concerns, including psychiatric symptoms, due to the absence of full-length dystrophin (Dp427), frequently accompanied by deficiencies in shorter isoforms. The lack of dystrophin affects neurophysiological processes from the uterine phase, impacting neural circuitry in brain regions such as the prefrontal cortex, hippocampus, and cerebellum. This leads to reduced inhibitory GABAergic transmission and altered hippocampal glutamatergic signaling. The resulting imbalance between inhibitory and excitatory inputs contributes to the neurodevelopmental and cognitive deficits observed in DMD. Recent studies have reported correlations between serum levels of D-aspartate and D-serine, endogenous ligands of glutamatergic receptors, and conditions such as schizophrenia, spinal muscular atrophy, and aging. Furthermore, in a recent clinical study, we reported a general dysregulation of D-/L-amino acids known to modulate glutamatergic neurotransmission in the serum of DMD patients, with significant correlations between muscle wasting, motor impairment, and alterations in L-glutamate levels and the L-glutamine/L-glutamate ratio. To delve deeper into this matter, we conducted an extensive neurochemical analysis using high-pressure liquid chromatography to measure the levels of the same D-/L-amino acids across various brain regions, the spinal cord, and serum of the mdx mouse model of DMD. Our results revealed a significant reduction in prenatal D-aspartate levels and postnatal levels of specific L-amino acids in the hippocampus of dystrophic mice compared to wild type. In adult mdx mice, we also observed a near-significant decrease in hippocampal D-serine levels and a significant reduction in spinal cord D-aspartate levels. This study provides the first evidence potentially linking D-/L-amino acid dysmetabolism in the hippocampus to the described neurophysiological alterations. Although further investigations are essential to validate this hypothesis, the mechanisms proposed here offer insight into how amino acid imbalances may contribute to the DMD-associated neurological and cognitive deficits, thus supporting the rationale for developing future targeted therapeutic strategies.

Abstract: NMDAR antagonists, such as memantine and ketamine, have shown efficacy in treating neurodegenerative diseases and major depression. The mechanism by which these drugs correct the aforementioned diseases is still unknown. Our study reveals that these antagonists significantly enhance 20S proteasome activity, crucial for degrading intrinsically disordered, oxidatively damaged, or misfolded proteins, factors pivotal in neurodegenerative diseases like Alzheimer's and Parkinson's. In our mouse model experiment, ketamine administration notably altered brain synaptic protein profiles within two hours, significantly downregulating proteins strongly associated with Alzheimer's and Parkinson's diseases. Furthermore, the altered proteins exhibited enrichment in terms related to plasticity and potentiation, including retrograde endocannabinoid signaling—a pivotal pathway in both short- and long-term plasticity that may elucidate the long-lasting effects of ketamine in major depression. Via the ubiquitin-independent 20S proteasome pathway (UIPS), these drugs maintain cellular protein homeostasis, which is crucial as proteasome activity declines with age, leading to protein aggregation and disease symptoms. Therefore, these findings hold promise for new treatment options not only for brain diseases but also for other systemic conditions associated with unfolded or misfolded proteins.

Abstract: ABSTRACT Autism spectrum disorder (ASD) is a neurodevelopmental psychiatric condition linked to glutamatergic neurotransmission disruption. Although endogenous D‐serine and D‐aspartate modulate glutamatergic N‐methyl D‐aspartate receptor (NMDAR) activity, their involvement in ASD remains elusive. We measured the levels of D‐aspartate, D‐serine, and other key neuroactive amino acids, and their direct precursors in brain regions, plasma, and feces of environmental ASD rat models prenatally exposed to lipopolysaccharide or valproate, both during adolescence and early adulthood, as well as in a genetic ASD model, the Fmr1‐ Δ exon8 rat. No significant changes were found in plasma and feces. Conversely, we observed a prominent accumulation of D‐aspartate in several brain regions of lipopolysaccharide‐ and valproate‐exposed rats, selectively during adolescence, while D‐serine level variations were more limited. No significant amino acid changes were observed in the Fmr1‐ Δ exon8 rat brain. We also assayed the activity of the main enzymes involved in cerebral D‐serine and D‐aspartate metabolism, suggesting that their regulation extends beyond their metabolic enzymes. These findings highlight that prenatal environmental stressors disrupt D‐amino acid levels selectively in ASD rat brains, emphasizing the role of early NMDAR dysfunction in ASD‐related phenotypes. image