Abstract Proton (1H) magnetic resonance spectroscopy (MRS) is a non‐invasive imaging technique that can be used to assess brain metabolism. Establishing reference levels of fetal brain metabolites in a translatable preclinical model is crucial for identifying subtle deviations from normal cerebral development. Herein, we developed a 1H‐MRS protocol for fetal sheep across three gestational periods (109 ± 2, 120 ± 5 and 139 ± 2 days gestation; term is 150 days gestation). Using this protocol, we established reference metabolic levels using recommended 1H‐MRS data analysis techniques. Objectives included adapting protocols for reliable metabolite detection despite the challenges of fetal 1H‐MRS, applying the protocol at multiple gestational ages, using recommended processing techniques and comparing two analysis software packages for robustness. MRI scans were performed on a 3 T Siemens clinical system (Magnetom Skyra, Siemens Healthineers, Germany) while the ewe was ventilated. 1H‐MRS was performed using a point‐resolved spectroscopy sequence at an echo time of 135 ms and a voxel size of 15 mm × 15 mm × 15 mm, gated to maternal respiration. We showed that TARQUIN metabolite level estimates had a significantly higher Cramér–Rao lower bound uncertainty compared with LCModel in N‐acetyl aspartate and choline, with differences in fit quality, while also underestimating metabolites. LCModel was then used to determine reference levels for N‐acetyl aspartate, choline, creatine and lactate across three gestational time points in late gestation. Our findings demonstrate the feasibility and reproducibility of non‐invasive fetal sheep brain 1H‐MRS. The study highlights the importance of objective criteria for accurate data interpretation, providing insights into fetal brain development and potential non‐invasive applications for earlier detection of poor neurodevelopmental outcomes.

STUDY QUESTION:Is prenatal maternal stress (PNMS) in humans associated with alterations in the sexually differentiated reproductive axis structures in the offspring? SUMMARY ANSWER:PNMS, experienced by pregnant mothers exposed to a natural disaster in 1998, was associated with structural changes in hypothalamic-pituitary-axis (HPG) structures of 18.5-year-old offspring. WHAT IS KNOWN ALREADY:In a human prospective study, PNMS was associated with higher childhood BMI, which in turn predicted earlier menarche. Epidemiological studies have shown that maternal life event stress is associated with changes in gonadal morphology and function. STUDY DESIGN, SIZE, DURATION:A prospective study of mothers exposed to a severe ice storm in 1998 (N = 224) collected measures of PNMS (objective hardship and subjective distress). Offspring reproductive structures were assessed in early adulthood. PARTICIPANTS/MATERIALS, SETTING, METHODS:PNMS-exposed offspring (ICE; n = 39, 21 F, 18 M) and a control group (n = 31, 14 F, 17 M; born in 1997) were assessed at age 18.5 years. Participants underwent MRI. Using a novel approach, the structural integrity of the entire HPG axis was assessed using gold-standard manual delineation from their MRI scans. Ovarian antral follicles (2-10 mm) were also counted from MRI images. Salivary estradiol and testosterone were measured with ELISA. Data were analyzed with ANOVA and multiple regression. MAIN RESULTS AND THE ROLE OF CHANCE:The expected sex difference in hypothalamic volume (M > F) was detected in controls. Within ICE, the sex difference was observed at low, but not at high, levels of PNMS (objective and subjective). Greater objective hardship was associated with smaller hypothalamic volume in men. Similarly, pituitary volume was smaller in ICE men compared to control men. Within ICE, greater objective hardship was associated with smaller left testicular volume. Finally, compared to controls, the left ovary had more antral follicles and tended to be larger in ICE women. The group differences and associations between PNMS and HPG morphology were generally medium to large. LIMITATIONS, REASONS FOR CAUTION:The relatively small sample size may have limited our ability to detect small differences and to test interactions with gestational timing of prenatal stress exposure. In addition, the majority of women were taking oral contraceptives, and thus results must be interpreted with caution. WIDER IMPLICATIONS OF THE FINDINGS:PNMS from a natural disaster may lead to functional changes in the human reproductive axis in adulthood. STUDY FUNDING/COMPETING INTEREST(S):Funding was provided by a grant from the Canadian Institutes of Health Research (CIHR, FRN 125892; to S.K., D.P.L., and J.C.P., and CIHR MOP 125892 to co-investigators S.K. S.L.J. was funded by a postdoctoral fellowship provided by the Fonds de Québec-Santé (FRQ-S). The funding sources were not involved in the collection, analyses, or interpretation of the data, or writing of the report. The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER:N/A.

Background: Altered excitation–inhibition (E/I) balance has been implicated in the pathophysiology of major depressive disorder (MDD), but previous magnetic resonance spectroscopy (MRS) findings have been inconsistent, partly due to methodological limitations. 7T MRS enables simultaneous quantification of glutamate (Glu) and γ-aminobutyric acid (GABA), and calculation of their ratio as an MRS-based proxy of E/I balance. Methods: Using 7T MRS, we measured Glu and GABA levels in the dorsal anterior cingulate cortex (dACC) of 41 treated patients with MDD and 35 matched healthy controls (HC). The primary outcome was the Glu/GABA ratio. Analysis of covariance tested group effects controlling for age and sex. Associations with clinical symptoms (Hamilton Depression Rating Scale) and cognitive performance were also evaluated. Results: A significant group × age interaction was observed (F(1, 57) = 8.49, p = 0.005), with Glu/GABA increasing with age in HC but not in MDD. No significant group differences were detected for Glu or GABA individually, although GABA levels showed a non-significant trend towards higher values in MDD. The Glu/GABA ratio was not associated with symptom severity or cognitive performance. Glu and GABA levels were positively correlated in both groups (MDD: r = 0.669, p < 0.001; HC: r = 0.513, p = 0.003), with no significant difference between the slopes. Conclusions: Treated patients with MDD exhibited an altered age-related trajectory of the Glu/GABA ratio in the dACC, characterized by a reduced Glu/GABA ratio and absence of the normative age-related increase, despite preserved Glu–GABA coupling.

Brain metabolism is vital to healthy brain function and is often altered in disease; yet direct investigation in patients is challenging. Although animal models are commonly used for studying brain metabolism, their use is under increasing scrutiny due to concerns of animal welfare and model validity. Human pluripotent stem cell (hPSC)-derived cerebral organoids (COs) present a unique opportunity to model human brain developmental and neuropathological processes, allowing for detailed metabolic characterization via multiple approaches. Here, we applied high-resolution magic-angle spinning (HR-MAS) proton nuclear magnetic resonance (1H-NMR) spectroscopy to analyze metabolite levels in hPSC-derived COs, establishing a pipeline to study neurometabolic pathways in these engineered human brain tissues. We identified and quantified 17 metabolites in hPSC-derived COs at different stages of maturity. The high spectral quality (linewidth < 4 Hz, SNR > 65) allowed detection of metabolite levels in 85- to 312-day-old hPSC-derived COs, which exhibited a metabolic profile similar to human fetal brain, with key distinguishing features relative to human adult brain, including: elevated lactate levels; approximately equimolar glutamate and glutamine levels; low N-acetylaspartate levels; and an abundance of hypotaurine. In summary, this study presents direct metabolic assessment in intact COs via HR-MAS 1H-NMR spectroscopy. Our approach provides a platform for investigating human brain metabolism and its alteration in human brain models of neurodegeneration.

Abstract Objective Reliable prognostic biomarkers in depression remain elusive. The objective of this study was to assess whether interleaved intermittent theta-burst stimulation-fMRI (iTBS-fMRI) can measure real-time target engagement, and prospectively test whether target engagement was associated with subsequent response to accelerated, connectivity-guided iTBS in treatment-resistant depression. Methods This single-centre trial ( NCT05813093 ) included 71 patients (42 with treatment-resistant depression [TRD], 29 ultra-treatment-resistant [UTRD]) who received personalized left dorsolateral prefrontal cortex (dlPFC) targeting based on functional connectivity with the subgenual anterior cingulate cortex (sgACC). Participants underwent one sham-controlled iTBS-fMRI session followed by five days of open-label accelerated iTBS (40 sessions; 600 pulses each). The primary outcome was change in HAM-D 17 . Primary imaging analyses examined active-sham interleaved iTBS-fMRI effects in the sgACC and left dlPFC regions, and their relationship with clinical outcomes. Results Accelerated iTBS was associated with rapid, sustained symptom reductions (ΔHAM-D 17 -9.01, p<0.001); overall response and remission rates were 40.8% (TRD: 47.6%, UTRD: 31.0%) and 16.9% (TRD: 16.7%, UTRD: 17.2%), with benefit maintained at 4 and 12 weeks. During interleaved iTBS-fMRI, active stimulation elicited significant target engagement within the sgACC-associated seedmap used for targeting (p=0.029). This brain response correlated with subsequent clinical improvement in depression and anxiety, but not other symptoms, and provided additional prognostic value independent of established clinical and demographic predictors. Conclusions Connectivity-guided accelerated iTBS was associated with rapid antidepressant effects in both TRD and UTRD participants with less pronounced effects in UTRD. Online target engagement measured by interleaved iTBS-fMRI is a candidate mechanistic biomarker that may aid prognosis in the future.

Alzheimer's disease (AD) is characterized by the appearance of brain pathology decades prior to clinical symptoms. The pre-symptomatic phase of AD provides opportunity for early detection and intervention. One early intervention that has been proposed is the use of non-steroidal anti-inflammatory drugs (NSAIDs), such as naproxen. However, evidence suggests that effects of naproxen intervention differ with stage of the disease, and the optimal intervention time is not clear. Accordingly, in this study, we investigated the impact of the timing of naproxen treatment in a rat model of AD. We used the TgF344-AD rat model of AD which develops characteristic pathological features of human AD, including abundant amyloid plaque pathology, astrogliosis, and microgliosis by 6 months of age, and neurofibrillary tangles and neuronal loss by 16 months of age. We examined the effects of naproxen treatment beginning at 1, 4, and 10 months of age in transgenic (Tg) animals and their wild-type (WT) littermates. We used longitudinal in vivo magnetic resonance spectroscopy (MRS) to study the impact of naproxen treatment on hippocampus neurochemistry. Previous studies have used MRS to non-invasively characterize the trajectory of altered hippocampal neurochemistry across the lifespan in the TgF344-AD rat model. In the current study, we employed MRS at 4, 10, and 16 months, i.e. prior to or after the appearance of amyloidosis and gliosis (6 months) and tau pathology (16 months), respectively, in Tg animals. Naproxen treatment altered neurochemistry in Tg animals only if administered beginning at 1 or 4 months of age, mitigating an otherwise observed increase in total choline and decrease in taurine. A more subtle effect was observed on the otherwise-expected increase in myo-inositol. These results highlight the possibility that earlier naproxen intervention could have distinct neurochemical effects compared to delayed treatment, though mechanistic implications remain to be clarified. Moreover, these findings support the use of MRS as a useful non-invasive method of monitoring treatment-related changes in neurochemistry in transgenic animal models.

PURPOSE:Quantification of metabolite concentrations using MRS requires tissue-dependent signal corrections. Accurate estimation of voxel tissue composition is therefore essential. Commonly used brain tissue segmentation tools differ in their algorithms and implementation, potentially introducing variability in MRS-derived concentration estimates. This study investigates the impact and reliability of tissue segmentation on metabolite quantification. METHODS:Three segmentation tools (ANTs, FSL, SPM) were evaluated using an in vivo test-retest MRI/MRS dataset. Voxelwise GM/WM/CSF fractions were applied to compute tissue-corrected total creatine (tCr) concentrations. Linear mixed-effects modeling, variance-component partitioning, and intraclass correlation coefficients (ICCs) quantified tool-, session-, and participant-related variance under permutation scenarios that isolated segmentation- and MRS-related effects. As a benchmark for segmentation performance, comparisons with manually segmented data were conducted across three brain regions. RESULTS:Segmentation tools produced systematically different tissue fractions that propagated into differences in tCr concentration estimates. Variance partitioning attributed 56.8%, 50.0%, and 51.3% of total tCr concentration variability to segmentation tool across the three permutations, with participant-specific factors accounting for 34.7%, 36.2%, and 28.5%, respectively. When segmentation variability was held constant, test-retest reliability was high (ICC > 0.8) but dropped to ∼0.5 when both segmentation and MRS variability varied. Agreement with manual segmentation was region- and tool-dependent, with the lowest agreement in the thalamus. CONCLUSION:Tissue segmentation contributes substantially to the variability in MRS-derived metabolite concentration estimates. These results underscore the need for transparent segmentation reporting and data sharing to ensure reproducibility and cross-study comparability in MRS research.

Glutamatergic changes are one of the characteristic features of psychotic disorders, with changes reported in their clinical high-risk states (CHR), first episode psychosis (FEP), and schizophrenia. Despite the abundance of literature using proton magnetic resonance (1H-MRS) to quantify glutamate, the general pattern of changes across different brain regions is not well studied. We investigated the presence of diagnosis group effects on glutamate concentrations in 99 participants in the left dorsal lateral prefrontal cortex, anterior cingulate cortex, associative striatum, and hippocampus using 1H-MRS in individuals with CHR (n = 31), schizophrenia spectrum psychotic disorder individuals (SCZ) with minimal antipsychotic exposure (n = 30), and healthy controls (n = 38). 1H-MRS data were normalized against white matter, gray matter, and cerebrospinal fluid fractions within regions of interest (ROI) to quantify local glutamate concentration. Our results showed no significant group differences in glutamate concentration across the four ROIs. Exploratory analyses showed a significant relationship between region and positive symptom severity on glutamate concentration. Post hoc regression analyses revealed that higher positive symptom severity was significantly associated with higher hippocampal glutamate concentrations. We conclude that glutamate concentrations may be related to positive symptoms.

Introduction- Choroid plexus (CP) enlargement on brain MRI has been identified as an emerging neuroinflammatory biomarker in multiple sclerosis (MS), yet its relationship to downstream parenchymal neurochemical abnormalities remains unknown. Proton magnetic resonance spectroscopy (1H MRS) enables non-invasive in vivo quantification of neurometabolites, making it well-suited to probe downstream consequences of CP pathology in MS. Methods- Ultra-high-field 7T 1H MRS was performed in 45 people with MS (pwMS) (28 Relapsing Remitting MS, RRMS; 17 Progressive MS, PMS) and 43 age- and sex-matched healthy controls (HCs) in the posterior cingulate cortex (PCC) and centrum semiovale white matter (CSWM). CP volume, EDSS, and MS Functional Composite measures were also acquired. Group differences in metabolite concentrations were evaluated using Mann-Whitney U tests with correction for multiple comparisons, and associations between CP volume, altered metabolites, and clinical disability and functional measures were investigated. Results- Myo-inositol (mI) was significantly elevated and total N-acetylaspartate was reduced in both MS subtypes, in the CSWM. In PMS, CP volume was positively associated with CSWM mI/total creatine (tCr) (ρ = 0.63, p = 0.008), an association absent in RRMS. Across the combined MS cohort, CP volume correlated significantly with EDSS (ρ = 0.40, p = 0.006). Conclusions- WM mI/tCr was elevated and tNAA/tCr was reduced across MS phenotypes compared with controls, reflecting a dual metabolic signature consistent with concurrent glial overactivation and neuroaxonal compromise. Increased CP volume was associated with greater neurological disability across MS phenotypes. The association of CP enlargement with CSWM mI/tCr in PMS suggests a potential link between CP-mediated periventricular inflammation and progressive WM glial pathology. Collectively, these findings support CP volume as a clinically relevant, non-invasive biomarker and restoring CP integrity as a potential therapeutic target in PMS, where effective treatments remain limited. ### Competing Interest Statement SS, FED, SA, JN and AB have nothing to report. DLA reports consulting fees from Biogen, Biohaven, BMS, Eli Lilly, EMD Serono, Find Therapeutics, Frequency Therapeutics, GSK, Idorsia Pharmaceuticals, Kiniksa Pharmaceuticals, Merck, Novartis, Race to Erase MS, Roche, Sanofi-Aventis, Shionogi, and Xfacto Communications; as well as an equity interest in NeuroRx, a Clario company. SN has received research funding from the Canadian Institutes of Health Research, the National Institutes of Health, the Myelin Repair Foundation, Immunotec, and F. Hoffman LaRoche; he has been a consultant for Sana Biotech and is a part-time employee of NeuroRx Research, a Clario Company. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study was approved by the Research Ethics Board of the McGill University Health Centre, the McGill University Institutional Review Board, Faculty of Medicine and Health Sciences, and the Aging-Neuroimaging Research Ethics Committee of the CIUSSS du Centre-Sud-de-île-de-Montréal (CIUSSS-CSMTL). Written informed consent was obtained from all participants. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors. Canadian Institutes of Health Research, https://ror.org/01gavpb45, 153005 Réseau en Bio-Imagerie du Quebec, https://ror.org/0271st670 Projets de Recherche Stratégiques et Structurants Fonds de Recherche du Québec - Santé, https://ror.org/02eqrsj93, 352199, 332672 Fondation Courtois

Magnetic resonance spectroscopic imaging (MRSI) is a non-invasive technique for probing metabolism. MRSI enables spatial mapping of metabolite distributions, offering insights into regional metabolic heterogeneity that single-voxel spectroscopy (SVS) cannot capture. However, MRSI produces large multidimensional datasets and requires complex processing pipelines, limiting reproducibility and accessibility. While human studies benefit from advanced processing tools, similar developments in preclinical research remain scarce, highlighting a demand for practical tools accessible to non-experts. Furthermore, recent advances in deuterium-based MRSI have opened metabolic pathway studies, introducing additional dimensions for kinetic information and specific positional labeling, thus substantially increasing dataset complexity and analysis demands. To address these needs, we introduce the MRS4Brain Toolbox, a freely available MATLAB-based platform for preclinical MRSI supporting proton, deuterium, and other nuclei, with extended functionalities for SVS and diffusion-weighted spectroscopy. The toolbox integrates reconstruction, preprocessing, quantification, quality control, brain segmentation automatically overlaid on metabolite maps, modeling, and statistical analysis into unified workflows accessible via a graphical interface. By streamlining data processing and reducing technical barriers, MRS4Brain Toolbox promotes reproducibility, harmonization, and broader adoption of basic and advanced spectroscopic techniques in preclinical studies, ultimately facilitating translational metabolic research.

INTRODUCTION:Oxidative stress may contribute to brain injury in the Alzheimer's disease (AD) continuum. The antioxidant glutathione (GSH) can be assessed with magnetic resonance spectroscopy (MRS). Because the relationship between GSH and vascular brain injury is unknown in the AD continuum, we address this gap in mild cognitive impairment (MCI). METHODS:3T Magnetic resonance imaging (MRI)/MRS data were obtained from 31 patients with MCI. GSH and total N-acetylaspartate (tNAA; neuroaxonal integrity marker) were measured in posterior cingulate cortex (PCC) and frontal white matter (FWM). Cerebrovascular injury was assessed using white matter hyperintensity (WMH) volume. Global and regional brain tissue integrity were assessed using normalized brain (NBV) and hippocampal volumes. RESULTS:Significant associations were reported in FWM between GSH/total creatine (tCr) and tNAA/tCr, and between GSH and both WMH and NBV. tNAA, GSH/tCr, and tNAA/tCr were higher in PCC than in FWM. DISCUSSION:Our results suggest that oxidative stress contributes to vascular brain injury in MCI. HIGHLIGHTS:Neuronal, vascular, and oxidative injuries occur in the Alzheimer's disease (AD) spectrum. Glutathione (GSH) is the main endogenous antioxidant in the brain. Brain GSH can be measured with magnetic resonance spectroscopy (MRS). We measured brain GSH level in people with mild cognitive impairment (MCI). Low GSH level was associated with vascular brain injury, neuroaxonal damage, and atrophy.

Magnetic resonance spectroscopy is a non-invasive technique for probing metabolism and underpins advanced methods such as magnetic resonance spectroscopic imaging (MRSI) and diffusion-weighted spectroscopy (DWS). MRSI enables spatial mapping of metabolite distributions, offering insights into regional metabolic heterogeneity that single-voxel spectroscopy (SVS) cannot capture. However, MRSI produces large multidimensional datasets and requires complex processing pipelines, limiting reproducibility and accessibility. While human studies benefit from advanced processing tools, similar developments in preclinical research remain scarce, highlighting a demand for practical tools accessible to non-experts. To address this need, we introduce the MRS4Brain Toolbox, a freely available MATLAB-based platform for preclinical spectroscopy, including MRSI, SVS, and DWS. The toolbox integrates reconstruction, preprocessing, quantification, quality control, brain segmentation automatically overlaid on metabolite maps, modeling, and statistical analysis into unified workflows accessible via a graphical interface. By streamlining data processing and reducing technical barriers, MRS4Brain Toolbox promotes reproducibility, harmonization, and broader adoption of advanced spectroscopic techniques in preclinical studies, ultimately facilitating translational research. ### Competing Interest Statement The authors have declared no competing interest. Swiss National Science Foundation, https://ror.org/00yjd3n13, project no. 201218, 10000465, 10006046, and 207935

OBJECTIVE:Interleaved TMS-fMRI has advanced understanding of network modulation but is limited to hemodynamic measures. We introduce a novel interleaved TMS-MRS platform, using standard MRI hardware, to assess real-time neurochemical changes and demonstrate feasibility in a clinical sample of patients with treatment-resistant depression (TRD). METHODS:Nine TRD participants (mean age 47.3 ± 13.9) underwent interleaved TMS-MRS on a 3T Siemens Prisma. A 20 mm3 voxel was placed over individualized left dlPFC targets. 1H-MRS spectra were acquired at baseline and during 10 Hz burst stimulation. Spectral quality and metabolite concentrations were compared across conditions. RESULTS:Spectral quality was preserved across conditions (FWHM baseline: 0.041 ± 0.005; active: 0.040 ± 0.005; p = 0.699) and signal-to-noise remained stable (baseline: 38.29 ± 5.38; active: 34.67 ± 9.12; p = 0.790). Two metabolites differed significantly: alanine increased during stimulation (0.58 ± 0.16 vs. 0.46 ± 0.14; p = 0.031), while NAA + NAAG decreased (9.07 ± 2.02 vs. 8.83 ± 0.99; p = 0.031). Exploratory analyses suggested associations between baseline and stimulation-induced metabolites (e.g., GSH, GABA, lactate) and clinical improvement following accelerated iTBS. CONCLUSION:Interleaved TMS-MRS is feasible with standard MRI hardware in TRD patients, enabling in-vivo detection of acute neurochemical changes during stimulation. This method offers a new avenue for probing excitatory/inhibitory balance, neuronal metabolism, and treatment mechanisms.

Magnetic resonance spectroscopy (MRS) offers a non-invasive modality to explore metabolic changes associated with Alzheimer’s disease (AD) pathogenesis and treatment efficacy. Studies in both humans and animal models have observed elevated choline in AD which may suggest an increase in cell membrane breakdown by phospholipase A2 (PLA2). PLA2 has also been linked to multiple hallmarks of AD pathology such as cognitive decline and inflammation (Figure 1). We hypothesized that PLA2 inhibition will reduce elevated choline levels and improve AD pathology. TgF344-AD rats (Tg) and non-transgenic (NTg) litter mates at 14 months old (N=50) were treated via daily IP injections with either 5mg/kg mepacrine (a global PLA2 inhibitor) or saline for one week followed by injections every other day for two weeks. Metabolite concentrations were measured in the right dorsal hippocampus (HC) and pre-frontal cortex (PFC) using localized proton MRS (Figure 2A) at baseline, 7 days, and 21 days. After 7 days of treatment, animals completed the Barnes maze including acquisition training, long-term memory probe (Figure 3A), and reversal training. At day 21, animals were sacrificed, tissues were collected, and brain sections were stained with 6F3D, PHF1, IBA1, and GFAP to observe changes to hippocampal pathology with PLA2 inhibition. At baseline, choline concentration was significantly elevated in the HC (6.8±1.9% increase, P=0.0012) but not the PFC (3.4±2.3% increase, P=0.1426; Figure 2B) of Tg animals. These levels were significantly reduced at day 7 and 21 of treatment in the HC of mepacrine treated Tg animals (6.6±1.9% decrease, P=0.0141; 6.9±1.3% decrease, P=0.0012; Figure 2B). In the PFC of mepacrine treated Tg animals, choline levels were only significantly reduced by day 21 of treatment (8.1±1.8% decrease, P=0.0027; Figure 2B). Contrary to our hypothesis, no significant differences were observed between saline and mepacrine treated Tg animals in any of the parameters measured in the probe trail (Figure 3B-F). Pathology data is currently being analyzed. These findings indicate choline concentration is associated with PLA2 activity in AD. However, changes to spatial memory formation with PLA2 inhibition were not observed. This is contrary to the literature; thus, further research is needed to understand if PLA2 plays a role in AD associated memory impairment.

BACKGROUND:Proton magnetic resonance spectroscopy (MRS) can be used to quantify multiple neurometabolites. However, due to the difficulty of separating overlapping signals at the commonly used field strength of 3 T, the quantified values are often composites of metabolically related chemicals. This can complicate interpretation and mask effects of interest. Therefore, it is important to determine the ability to accurately separate these signals at 3 T. Data acquired at 7 T can provide a benchmark, as higher field strength facilitates spectral resolution and reduces the signal overlap. NEW METHODS:This study assessed the ability of multiple 3 T MRS sequences to separate the commonly acquired neurochemicals (Glutamate (Glu) and Glutamine (Gln); N-Acetyl aspartate (NAA) and N-acetylaspartylglutamate (NAAG); Creatine (Cr) and Phosphocreatine (PCr); Choline (Cho), Phosphocholine (PC) and Glycerophosphocholine (GPC)). We compared metabolites quantified at 3 T from 6 acquisitions (PRESS, TE= 20, 30, 40, 80 ms, semi-LASER, TE=28 ms and STEAM TE=6 ms) with those quantified at 7 T using STEAM (TE=8 ms). RESULTS:Sequences with short echo times (STEAM-6, PRESS-20) generally performed better at separating most metabolites when using correlative and difference analyses with 7 T reference data. The exceptions were NAAG, which was best quantified with PRESS-80, and Cr and PCr, which were not well separated by any sequence. COMPARISON WITH EXISTING METHODS AND CONCLUSION:When wanting to specifically separate composite metabolite signals using single voxel MRS, shorter echo times generally perform better. Researchers should be mindful of the effects of acquisition parameters on the metabolite measures.

BACKGROUND:Magnetic resonance spectroscopy (MRS) offers a non-invasive modality to explore metabolic changes associated with Alzheimer's disease (AD) pathogenesis and treatment efficacy. Studies in both humans and animal models have observed elevated choline in AD which may suggest an increase in cell membrane breakdown by phospholipase A2 (PLA2). PLA2 has also been linked to multiple hallmarks of AD pathology such as cognitive decline and inflammation (Figure 1). We hypothesized that PLA2 inhibition will reduce elevated choline levels and improve AD pathology. METHOD:TgF344-AD rats (Tg) and non-transgenic (NTg) litter mates at 14 months old (N=50) were treated via daily IP injections with either 5mg/kg mepacrine (a global PLA2 inhibitor) or saline for one week followed by injections every other day for two weeks. Metabolite concentrations were measured in the right dorsal hippocampus (HC) and pre-frontal cortex (PFC) using localized proton MRS (Figure 2A) at baseline, 7 days, and 21 days. After 7 days of treatment, animals completed the Barnes maze including acquisition training, long-term memory probe (Figure 3A), and reversal training. At day 21, animals were sacrificed, tissues were collected, and brain sections were stained with 6F3D, PHF1, IBA1, and GFAP to observe changes to hippocampal pathology with PLA2 inhibition. RESULT:At baseline, choline concentration was significantly elevated in the HC (6.8±1.9% increase, P=0.0012) but not the PFC (3.4±2.3% increase, P=0.1426; Figure 2B) of Tg animals. These levels were significantly reduced at day 7 and 21 of treatment in the HC of mepacrine treated Tg animals (6.6±1.9% decrease, P=0.0141; 6.9±1.3% decrease, P=0.0012; Figure 2B). In the PFC of mepacrine treated Tg animals, choline levels were only significantly reduced by day 21 of treatment (8.1±1.8% decrease, P=0.0027; Figure 2B). Contrary to our hypothesis, no significant differences were observed between saline and mepacrine treated Tg animals in any of the parameters measured in the probe trail (Figure 3B-F). Pathology data is currently being analyzed. CONCLUSION:These findings indicate choline concentration is associated with PLA2 activity in AD. However, changes to spatial memory formation with PLA2 inhibition were not observed. This is contrary to the literature; thus, further research is needed to understand if PLA2 plays a role in AD associated memory impairment.

Alterations in excitatory neurotransmitters, involving the glutamate (Glu) and glutamine (Gln) cycle, as well as inhibitory neurotransmission, GABA, are implicated in the pathophysiology of autism spectrum disorder (ASD). Although magnetic resonance spectroscopy (MRS) holds promise for assessing these metabolites, conventional 3 T MRI does not robustly measure them, leaving the neurochemical pathophysiology of ASD insufficiently understood. 7 T MRI enables reliable assessments of these neurometabolites by enhancing the signal-to-noise ratio and improving the spectral resolution, particularly in distinguishing neuroactive Glu from its metabolic precursor, Gln. The current 7 T MRS study has two primary objectives: first, to investigate neurometabolite levels in adults with ASD to elucidate its neurochemical pathophysiology, and second, to examine their association with symptoms of ASD. Thirty-three adults with ASD (mean age = 31 years) and 52 age-matched control adults were included. The neurometabolite levels of Glu, Gln, and GABA were assessed in the anterior cingulate cortex (ACC), thalamus, and right temporo-parietal junction (TPJ), with most quantifications passing quality checks. Analysis of covariance revealed significant effects of diagnosis on Gln in the thalamus (p = 0.008) and right TPJ (p = 0.006), indicating elevated Gln levels in these regions in the ASD group. Among social communication and restricted and repetitive behaviors, significant negative correlations were observed in the ASD group between Gln levels and sensory symptoms. These findings suggest that alterations in the excitatory neurotransmission regulation, presumably increased cycling of the Gln-Glu circuit, may underlie the pathophysiology of ASD.

Effortful learning and practice are integral to academic attainment in areas like reading, language, and mathematics, shaping future career prospects, socioeconomic status, and health outcomes. However, academic learning outcomes often exhibit disparities, with initial cognitive advantages leading to further advantages (the Matthew effect). One of the areas in which learners frequently exhibit difficulties is mathematical learning. Neurobiological research has underscored the involvement of the dorsolateral prefrontal cortex (dlPFC), the posterior parietal cortex (PPC), and the hippocampus in mathematical learning. However, their causal contributions remain unclear. Moreover, recent findings have highlighted the potential role of excitation/inhibition (E/I) balance in neuroplasticity and learning. To deepen our understanding of the mechanisms driving mathematical learning, we employed a novel approach integrating double-blind excitatory neurostimulation-high-frequency transcranial random noise stimulation (tRNS)-and examined its effect at the behavioral, functional, and neurochemical levels. During a 5-day mathematical learning paradigm (n = 72) active tRNS was applied over the dlPFC or the PPC, and we compared the effects versus sham tRNS. Individuals exhibiting stronger positive baseline frontoparietal connectivity demonstrated greater improvement in calculation learning. Subsequently, utilizing tRNS to modulate frontoparietal connectivity, we found that participants with weaker positive baseline frontoparietal connectivity, typically associated with poorer learning performance, experienced enhanced learning outcomes following dlPFC-tRNS only. Further analyses revealed that dlPFC-tRNS improved learning outcomes for participants who showed reductions in dlPFC GABA when it was accompanied by a reduced positive frontoparietal connectivity, but this effect was reversed for participants who showed increased positive frontoparietal connectivity. Our multimodal approach elucidates the causal role of the dlPFC and frontoparietal network in a critical academic learning skill, shedding light on the interplay between functional connectivity and GABAergic modulation in the efficacy of brain-based interventions to augment learning outcomes, particularly benefiting individuals who would learn less optimally based on their neurobiological profile.

Proneural genes are conserved drivers of neurogenesis across the animal kingdom. How their functions have adapted to guide humanspecific neurodevelopmental features is poorly understood. Here, we mined transcriptomic data from human fetal cortices and generated from human embryonic stem cell-derived cortical organoids (COs) to show that NEUROG1 and NEUROG2 are most highly expressed in basal neural progenitor cells, with pseudotime trajectory analyses indicating that NEUROG1-derived lineages predominate early and NEUROG2 lineages later. Using ChIP-qPCR, gene silencing and overexpression studies in COs, we show that NEUROG2 is necessary and sufficient to directly transactivate known target genes (NEUROD1, EOMES, RND2). To identify new targets, we engineered NEUROG2-mCherry knock-in human embryonic stem cells for CO generation. The mCherry-high CO cell transcriptome is enriched in extracellular matrix-associated genes, and two genes associated with human-accelerated regions: PPP1R17 and FZD8. We show that NEUROG2 binds COL1A1, COL3A1 and PPP1R17 regulatory elements, and induces their ectopic expression in COs, although NEUROG2 is not required for this expression. Neurog2 similarly induces Col3a1 and Ppp1r17 in murine P19 cells. These data are consistent with a conservation of NEUROG2 function across mammalian species.