Astroglial disinhibition of cortical circuits disrupts cognition via kynurenic acid

Astroglial disinhibition of cortical circuits disrupts cognition via kynurenic acid | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Astroglial disinhibition of cortical circuits disrupts cognition via kynurenic acid Tina Notter, Viktor Beilmann, Johanna Furrer, Sina Schalbetter, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4492882/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Astrocytes play critical roles in neural circuit function, but how they contribute to cognitive impairment remains poorly understood. Here, we identify astrocyte-derived kynurenic acid (KYNA), a neuroactive metabolite acting as endogenous N-methyl-D-aspartate receptor (NMDA) receptor antagonist, as a key mediator of cognitive dysfunction in the context of aberrant astrocyte activity. Using chemogenetic stimulation, pharmacological rescue, and astrocyte-specific knockdown of kynurenine aminotransferase II (KAT II), we show that elevated KYNA suppresses parvalbumin-positive interneuron activity in the prefrontal cortex, leading to disinhibition of pyramidal neurons and impairments in cognitive functions linked to cortical activity, including episodic-like and working memory as well as sensorimotor gating. These findings define an astrocyte-KYNA-interneuron axis that controls cortical excitability and cognition, linking glial metabolism to circuit imbalance and cognitive dysfunction with broad relevance to psychiatric and neurological disorders. Biological sciences/Neuroscience/Glial biology/Astrocyte Biological sciences/Neuroscience/Diseases of the nervous system/Schizophrenia Biological sciences/Neuroscience/Diseases of the nervous system/Developmental disorders Astrocytes cognition kynurenine pathway maternal immune activation (MIA) prefrontal cortex parvalbumin interneuron Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Astrocytes are increasingly recognized as active participants in neural circuit dynamics. In addition to their classical roles in metabolic and homeostatic support, they modulate synaptic transmission, shape network activity, and influence behavioral states 1 , 2 . Astrocyte physiology is markedly altered in many neurological and psychiatric conditions 3 . Inflammatory and disease-associated environmental stimuli trigger changes in astrocytic gene expression, intracellular signaling, and metabolic output. These reactive states have been observed in disorders ranging from epilepsy and neurodegeneration to schizophrenia and depression 4 , 5 . Despite growing recognition that astrocytes are affected in central nervous system (CNS) disorders, the consequences of these altered states for circuit function and cognition remain poorly understood. This question is especially pertinent to the prefrontal cortex (PFC), a region central to higher-order cognitive function, where circuit dysregulation is strongly linked to cognitive deficits in psychiatric disorders 6 . Cortical circuits in the PFC depend on the precise balance of excitatory and inhibitory activity to sustain working memory, attention, and sensorimotor integration 7 . Maintenance of this balance depends on the coordinated interaction between inhibitory interneurons and pyramidal cells, with fast-spiking parvalbumin-positive (PV+) interneurons and their NMDAR activity playing a key role 7 , 8 . However, whether astrocytes influence these circuits, and the molecular mechanisms by which altered astrocyte activity impacts prefrontal computation and behavior, are not well defined. RESULTS Astrocyte stimulation in the PFC elevates KYNA and alters cortical circuit activity To examine how altered astrocyte states impact PFC circuit function, we selectively increased astrocyte activity using chemogenetics. To this end, Gq-signaling was activated in PFC astrocytes of adult mice (Fig. 1 a-c). In vivo two-photon Ca²⁺ imaging in awake mice confirmed robust and repeatable clozapine- N -oxide (CNO)-induced Ca²⁺ elevation in transduced astrocytes (Fig. 1 d, Supplementary Fig. S1 ). Using this approach, we then examined whether altering PFC astrocyte states shifts the metabolic pathway of kynurenine (KYN) degradation. We focused on this pathway (Fig. 1 e) because its downstream metabolite, kynurenic acid (KYNA), is an endogenous NMDA receptor antagonist implicated in cognitive dysfunction and predominantly synthesized by astrocytes 9 , 10 . Notably, PFC KYNA levels and the KYNA/KYN ratio were significantly elevated in CNO-treated mice, while KYN levels and other pathway metabolites remained unchanged (Fig. 1 f, Supplementary Fig. S2 ). According to the excitatory-inhibitory (E/I) imbalance hypothesis, NMDA receptor antagonists, such as ketamine, disrupt prefrontal circuit function by suppressing parvalbumin-positive (PV+) interneuron activity, leading to disinhibition of pyramidal neurons and cognitive impairment 11 – 13 . Since KYNA is an endogenous NMDAR antagonist primarily produced by astrocytes 10 , 14 , we hypothesized that astrocyte-induced elevated KYNA may similarly reduce PV + interneuron activity and thereby shift prefrontal circuit dynamics toward excitation (Fig. 1 g). To test this, we combined in vivo two-photon Ca 2+ imaging in PV + interneurons (Fig. 1 h) with cell-type-specific c-Fos mapping (Fig. 1 i) following chemogenetic activation of prefrontal astrocytes. In line with our hypothesis, CNO treatment reduced PV + interneuron activity as evident by the reduction in spike activity (Fig. 1 h) and c-Fos expression in PV + interneurons (Fig. 1 j, Supplementary Fig. S3 a ), while increasing c-Fos in CaMKII + excitatory pyramidal neurons (Fig. 1 k, Supplementary Fig. S3 b ). No changes were observed in somatostatin-positive (SST+) interneurons ( Supplementary Fig. S3 c ), indicating a selective effect on PV + cells. Together, these findings demonstrate that altering PFC astrocyte states increases KYNA and suppresses PV + interneuron activity, leading to disinhibition of pyramidal neurons. This shift in circuit dynamics could provide the mechanistic basis through which astrocytes could contribute to cognitive impairment. Astrocyte stimulation impairs memory and pre-attentive filtering To assess whether astrocyte-induced changes in KYNA and circuit dynamic in the PFC translate into cognitive dysfunctions, we subjected mice to different behavioral and cognitive tests following CNO administration (Fig. 2 a). Chemogenetic activation of prefrontal astrocytes impaired performance across several PFC-dependent cognitive domains. In the temporal order memory test for episodic-like memory, CNO-treated male mice failed to discriminate object recency compared to vehicle (VEH) treatment (Fig. 2 b). In the Y-maze test for working memory, astrocyte stimulation reduced spontaneous alternation without affecting total entries (Fig. 2 c). These effects occurred without changes in anxiety-like behavior or locomotion ( Supplementary Fig. S4 ), demonstrating selective effects of astrocyte activation on cognitive performance. Furthermore, CNO-treatment led to the disruption of prepulse inhibition (PPI) of the acoustic startle reflex (Fig. 2 d), a form of pre-attentive filtering deficient in various psychiatric disorders 15 and impaired by elevated endogenous brain KYNA 16 . Control mice expressing a GFAP-driven EGFP construct showed no behavioral effects of CNO ( Supplementary Fig. S5 ), ruling out off-target drug effects. Similar deficits were observed in female hM3DGq mice ( Supplementary Fig. S6 ), showing that the astrocyte-induced cognitive disruption is present in male and female mice. Together, these findings demonstrate that selective stimulation of prefrontal astrocytes impairs cognitive functions and pre-attentive filtering. The shift towards increased KYNA may underlie the observed changes in circuit activity and behavior, warranting further testing of causality. KYNA mediates astrocyte-induced cognitive deficits via PV interneuron suppression To test whether elevated KYNA causally mediates the cognitive impairments and circuit alterations, we first pharmacologically inhibited its synthesis. We used PF-04859989, a brain-penetrant inhibitor of KAT II, which efficiently lowers brain KYNA levels after systemic administration 17 . Mice received PF-04859989 (1 mg/kg or 10 mg/kg) 2.5 hours before CNO-induced astrocyte activation (Fig. 3 a). While sub-threshold dose (1 mg/kg) had no effect, the higher dose (10 mg/kg) fully restored KYNA levels, temporal order memory, working memory, and PPI in CNO-treated mice (Fig. 3 b- 3 d, Supplementary Fig. S7a ). These effects were not due to general cognitive enhancement. When the same doses of PF-04859989 were administered to non-activated control mice, no significant changes in cognitive performance were observed ( Supplementary Fig. S7b-e ). Thus, KAT II inhibition selectively reversed the cognitive deficits induced by astrocyte activation, without baseline pro-cognitive effects. We next examined whether KYNA mediates the shift in cortical circuit dynamics. Administration of 10 mg/kg PF-04859989 prior to CNO treatment restored temporal order memory performance (Fig. 3 e) and normalized c-Fos levels in both PV + interneurons (Fig. 3 f, Supplementary Fig. S8a ) and CaMKII + pyramidal neurons (Fig. 3 g, Supplementary Fig. S8b ), without affecting SST + interneurons ( Supplementary Fig. S8c ). To further validate the role of astrocyte-derived KYNA in driving these dysfunctions, we next used a genetic approach to block KYNA synthesis selectively in activated prefrontal astrocytes. We hereby injected custom-made AAVs expressing four unique miRNA-adapted shRNAs targeting mAadat (referred to as KATII KD ) along with hM3DGq and an HA tag into the PFC of male mice (Fig. 4 a). This design enabled simultaneous chemogenetic activation and selective knockdown of KAT II in prefrontal astrocytes. Knockdown efficacy was confirmed by reduced mAadat mRNA and decreased protein levels of both the ~ 47 kDa KAT II monomer and its active ~ 94 kDa homodimer 18 in PFC homogenates of KATII KD mice compared to wild-type controls (Fig. 4 b, c). Immunohistochemistry confirmed astrocyte-specific expression in the PFC, as observed with the original hM3DGq-mCherry construct (Fig. 4 d). In vivo two-photon Ca²⁺ imaging in anesthetized mice demonstrated that hM3DGq-mediated astrocyte activation remained functional in the KATII KD condition, as indicated by CNO-induced Ca²⁺ elevations (Fig. 4 e). We next assessed whether reducing KAT II expression was sufficient to prevent astrocyte-induced cognitive dysfunction (Fig. 4 f). KAT II knockdown prevented the emergence of episodic-like memory (Fig. 4 g) and working memory deficits (Fig. 4 h), as well as the disruption of PPI of the acoustic startle reflex (Fig. 4 i). A concurrent normalization was observed on the level of PV + interneurons and pyramidal cell activity. Mice expressing KATII KD -hM3DGq-HA that failed to show any deficits in temporal order memory (Fig. 4 j), working memory ( Supplementary Fig. S9a ), or PPI ( Supplementary Fig. S9b ) upon CNO treatment, also displayed unchanged levels of c-Fos expression in both PV + and CaMKII + neurons compared to controls (Fig. 4 k, l; Supplementary Fig. S9c ). These findings confirm that circuit alterations and cognitive impairments were dependent on astrocyte-derived KYNA. Together, these findings identify an astrocyte-KYNA-interneuron axis, whereby astrocyte-derived KYNA impair cognition via impeding PFC E/I balance by selectively suppressing PV + interneuron activity, leading to disinhibition of pyramidal neurons. KYNA contributes to cognitive dysfunction in a neuropsychiatric risk model To examine how altered astrocyte states and kynurenine metabolism relate to cognitive dysfunction within a translationally relevant disease framework, we used a mouse model of maternal immune activation (MIA), an established environmental risk factor for neurodevelopmental disorders with cognitive impairments 19 , 20 . MIA was induced by prenatal administration of poly(I:C), a viral mimetic that triggers an acute inflammatory response during fetal development (Fig. 5 a) 21 . Consistent with previous findings 22 , MIA impaired the ability of offspring to discriminate objects based on the temporal presentation (Fig. 5 b), which emerged in the absence of concomitant changes in basal locomotor and innate anxiety-like behavior ( Supplementary Fig. S10 ). We next assessed whether MIA alters astrocyte states in the PFC. GFAP immunoreactivity was significantly elevated in MIA offspring, consistent with increased astrocyte reactivity (Fig. 5 c). While other astrocytic markers remained unchanged ( Supplementary Fig. S11 ), individual GFAP levels negatively correlated with cognitive performance in MIA but not control mice (Fig. 5 d), linking astrocyte reactivity to cognitive impairment. We then investigated whether these behavioral and glial changes were accompanied by alterations in kynurenine (KYN) metabolism. Compared to controls, MIA offspring displayed elevated plasma KYN levels, the blood brain barrier (BBB)-permeable and immediate precursor of central KYNA 16 , without changes in peripheral BBB-impermeable KYNA (Fig. 5 e). Notably, KYN levels negatively correlated with memory performance (Fig. 5 f) and positively with prefrontal GFAP intensity (Fig. 5 g), suggesting a coordinated relationship between systemic KYN, astrocyte reactivity, and behavioral deficits. To test whether elevated KYNA contributes causally to cognitive impairments, adult MIA and control offspring were treated with PF-04859989 (10 mg/kg) or vehicle before testing (Fig. 5 h). In MIA offspring, PF-04859989 restored temporal order memory to control levels (Fig. 5 i). This effect was not attributable to a general enhancement of cognitive performance, as PF-04859989 treatment had no impact in control offspring, indicating specificity to the pathophysiological state. Together, these findings link heightened prefrontal astrocyte reactivity and KYNA to impaired episodic-like memory performance in a mouse model of psychiatric disease risk, supporting an astrocyte-KYNA contribution to cognitive dysfunction under pathophysiological conditions. DISCUSSION Our study reveals a previously unrecognized astrocyte-interneuron signaling mechanism that shapes prefrontal circuit function and cognition. We demonstrate that increased astrocyte activity impairs cognitive performance by elevating the neuroactive metabolite KYNA, the brain’s only known endogenous NMDAR antagonist 14 , which selectively suppresses PV + interneuron activity and in turn disinhibits pyramidal neurons. The KYNA-driven excitatory-inhibitory imbalance and cognitive deficits are reminiscent of those induced by synthetic NMDAR antagonists such ketamine 11 , 12 , 17 , 23 , PCP 24 , or MK-801 25 . While prior work has implicated astrocyte-derived KYNA in the modulation of excitatory transmission 9 , our study establishes the first causal link between astrocyte-driven KYNA elevation, cell-type-specific neuronal effects, and cognitive impairment in vivo. Our findings complement recent work on astrocyte-mediated state transitions 26 , 27 by identifying a defined metabolic signaling axis (astrocyte-KYNA-interneuron) that disrupts local circuit balance and cognitive performance. Importantly, this axis engages PV + interneurons, a neuronal population consistently implicated in psychiatric disease pathophysiology 28 , 29 , and positions astrocytes as upstream modulators of their activity via KYNA. Concurrent to this mechanistic link, our data reinforce the relevance of the KYN pathway in psychiatric diseases. Elevation of prefrontal KYNA levels in the astrocyte DREADD model parallels findings in patients with schizophrenia or bipolar disorder, where increased KYNA has been detected in cortical or cerebrospinal fluid samples 30 – 34 . Although the exact sources of KYNA in human disease remain unclear, our results suggest that astrocytes are not only capable of producing KYNA under pathophysiological conditions, but also directly contribute to cognitive dysfunction when KYNA levels are elevated. We also demonstrate that astrocyte reactivity and KYN metabolism correlate with cognitive deficits in offspring subjected to MIA, a well-established neurodevelopmental model of psychiatric disease risk 19 , 20 . While MIA induces widespread changes in brain development and processes 20 , 21 , our data point to a prefrontal astrocyte-KYNA axis as a disease-relevant contributor to cognitive impairments. This is consistent with human studies linking elevated peripheral KYN levels to cognitive deficits in a subset of schizophrenia patients 34 . Moreover, pharmacological inhibition of KAT II, the enzyme converting KYN into KYNA, rescued memory and sensorimotor gating deficits in both MIA and DREADD models, without inducing pro-cognitive effects in control animals. These findings identify KAT II as a potential therapeutic target for conditions marked by glial dysregulation and cognitive dysfunction. While our study focused specifically on astrocyte activity in the PFC, the astrocyte-KYNA-interneuron signaling axis we define may operate in other brain regions and disease contexts as well. The ability of astrocytes to reshape local circuit dynamics via metabolic modulation of interneuron activity expands our understanding of glial contributions to behavior and identifies a mechanistic link between astrocyte reactivity, KYN metabolism, and cognitive dysfunction. Given the prominence of astrocyte dysregulation, elevated KYNA levels, and PV interneuron deficits across psychiatric disorders such as schizophrenia and bipolar disorder 9 , 28 , 30 , 35 – 37 , these findings provide a conceptual and mechanistic framework for targeting astrocyte-derived metabolites in the treatment of cognitive symptoms. MATERIAL AND METHODS Animals All experiments were performed using male or female C57BL6/N mice (Charles Rivers, Sulzfeld, Germany). They were group-housed (4–5 animals per cage) in individually ventilated cages under a reversed light–dark cycle. All animals had ad libitum access to standard rodent chow and water throughout the entire study. All procedures were conducted during the dark cycle and had been previously approved by the Cantonal Veterinarian’s Office of Zurich. All efforts were made to minimize the number of animals used and their suffering. Details about housing conditions and an overview of the different cohorts of animals and the respective numbers used are provided in the Supplementary Table 1 . DREADD system The DREADD system was based on a recombinant adeno-associated virus serotype 9 (AAV9) that expresses hM3DGq under the astrocyte-specific promoter hgfaABC1D (hGFAP), encompassing a fluorescent mCherry tag (AAV9-hGFAP-hM3DGq-mCherry; Fig. 1 a). Some experiments involved an EGFP-tagged control virus (ConV) with the same promoter (AAV9-hGFAP-EGFP) and the GCaMP6s Ca 2+ sensor (AAV9-hGFAP-GCaMP6s). In addition, some experiments involved a custom-made virus that expresses 4 unique miRNA-adapted shRNA hairpins directed against mAadat mRNA together with hM3DGq under the same hGFAP promoter (AAV9-hGFAP-chI[4x:sh(mAadat)]-HA-hM3DGq, herein referred to as AAV9-hGFAP-KATIIKD-hM3DGq-HA), see below and Supplementary Information ). All AAVs were produced and purchased from the Viral Vector Facility of the University of Zurich, Switzerland ( www.vvf.uzh.ch ) and were injected into the PFC using bilateral stereotaxic injections (stereotaxic coordinates: anteroposterior [AP] = + 1.8 mm, mediolateral [ML] = ± 0.3 mm, dorsoventral [DV] = − 1.9 mm) as described in the Supplementary Information . hM3DGq was activated with 1 mg/kg clozapine-N-oxide (CNO, BML-NS105-0025, Enzo Life Sciences, Switzerland) dissolvedac in 0.9% NaCl (B. Braun, Switzerland). The dose of 1 mg/kg was chosen based on previous chemogenetic studies in rodents 39 – 42 . Treatment with vehicle (VEH; 0.9% NaCl) served as control treatment. ConV-expressing mice receiving CNO or VEH were used to exclude non-selective effects of CNO 43 . CNO (1 mg/kg) or VEH were given via the micropipette-guided drug administration (MDA) method, a non-invasive oral administration technique described in detail elsewhere 42 , 44 . Experiments involving in vivo two-photon imaging in anesthetized mice VEH or CNO (1mg/kg) were injected s.c. with an injection volume of 2 mL/kg ( Supplementary Information ). Kynurenine aminotransferase II inhibition Kynurenine aminotransferase II (KAT II) was inhibited with 1 mg/kg or 10 mg/kg PF-04859989 hydrochloride (PZ0250, Sigma-Aldrich), which was dissolved in sterile water and freshly prepared prior to each experiment. For animals subjected to behavioral testing, 1 or 10 mg/kg PF-04859989, or sterile water (vehicle, 0 mg/kg) only, was injected i.p. using an injection volume of 5 mL/kg 3 hrs prior to each behavioral test. For postmortem analyses of c-Fos expression, sterile water (vehicle) or 10 mg/kg PF-04859989 was administered either 5 hours (behaviorally naïve animals) or 8 hrs (behaviorally tested animals) prior to tissue collection. The doses and post-injection interval were chosen based on previous dose-response studies in rodents 17 , 45 . Astrocyte selective knockdown of Kynurenine aminotransferase II inhibition. To selectively knockdown KAT II expression in astrocytes, we employed the miRNA-adapted short/small hairpin (sh) RNA (shRNAmir) strategy 46 , 47 . Construct design and AAV production were conducted by the Viral Vector Facility, University of Zurich, Switzerland according to established protocols. shRNA silencing constructs were custom designed against the mAadat transcript NM_011834.2. Four 21-mer shRNA sequences were selected based on their target selectivity and highest probability ranking for knockdown efficacy. The shRNA sequences were: 5’-GCAACAACCCTACAGGCAACT-3’, 5’-GGTTGAGAGTAGGGTTTATGA-3’, 5’-GGTTTATGACTGGCCCTAAGA-3’, and 5’-GGGTTTCCTGGCTCATATTGA-3’. An shRNAmir-E cassette containing the four shRNAs was cloned into a pssAAV-2-hGFAP-HA_hM3D(Gq)-bGHp(A) backbone, to generate the pssAAV-2-hGFAP-chI[4x:sh(mAadat)]-HA-hM3DGq-bGHp(A) plasmid. The construct was then packaged in AAV9 to produce the AAV9-hGFAP-chI[4x:sh(mAadat)]-HA-hM3DGq (referred to as AAV9-hGFAP-KATIIKD-hM3DGq-HA or short KATIIKD-hM3DGq), allowing for region and cell type-selective knockdown of KAT II expression. Knockdown efficacy was assessed 4 weeks after construct expression by real-time quantitative PCR (RT-qPCR) and Western blot analyses using total RNA and proteins extracted from PFC of male C57BL6/N mice expressing the KATIIKD-hM3DGq construct in prefrontal astrocytes or C57BL6/N control male mice expressing no construct (referred to as wild-type). Methodological details regarding RT-qPCR and Western blot experiments are available in the Supplementary Information . Maternal immune activation model Two independent cohorts of time-pregnant mice were used in this study. The cohorts were generated via on-site breeding under identical experimental conditions as described in detail in the Supplementary Information . Cohort 1 was used for the assessment of cognitive deficits, followed by post-mortem analyses (Fig. 1 ). Cohort 2 was used for the pharmacological rescue study of the cognitive deficits using the KATII inhibitor PF-04859989 (Fig. 3 ). On gestational day (GD) 12, pregnant mice were randomly assigned to a single injection of poly(I:C) (potassium salt, P9582, Sigma–Aldrich, Buchs, St. Gallen, Switzerland) or treatment with endotoxin-free 0.9% NaCl (B. Braun, Melsungen, Switzerland) vehicle solution. Male offspring to CON and MIA dams were weaned and housed in groups of 2 to 5 per cage as described above. Behavioral testing in both cohorts commenced when the offspring reached 12 weeks of age. A minimum of 1-week resting period was imposed after behavioral testing before the animals were killed and tissue was collected for subsequent post-mortem analyses (see below). Methodological details regarding the MIA model, including timed-mating, treatment, birth conditions and weaning of offspring, can be obtained in the Supplementary Information and the reporting guideline checklist for the MIA model 38 provided in Supplementary Table S2 . Behavioral and cognitive testing Behavioral and cognitive testing included temporal order memory test for objects, a spontaneous alternation task for working memory in the Y-maze, a prepulse inhibition (PPI) test of the acoustic startle reflex for pre-attentive filtering, as well as tests assessing locomotor activity and innate anxiety-like behavior (open field and light-dark box tests). Mice undergoing multiple behavioral tests were given a minimum inter-test interval (ITI) of 48 hours between tests with the exception between open field test and the temporal order memory test. Because the open field test also serves as habituation to the arena for the temporal order memory test, a 24-hour ITI was implemented between the two tests. For the hM3DGq-based DREADD model, 30 min prior to each behavioral test mice were treated once with VEH or CNO (1mg/kg) via MDA. A detailed description of the methodological procedures and rationale of inclusion for each behavioral and cognitive test are provided in the Supplementary Information . In vivo two-photon Ca 2+ imaging of prefrontal astrocytes in mice using microprisms In vivo Ca 2+ imaging using two-photon microscopy in awake or anesthetized mice was applied to ascertain the effectiveness and temporal dynamics of hM3DGq-based elevation of Ca 2+ in prefrontal astrocytes or Ca 2+ responses in PV interneurons. To this end, hM3DGq or KATII KD -hM3DGq expressing AAV was co-injected with an AAV expressing the Ca 2+ sensor, GCaMP6s (astrocytes) or loxP(rev)jGCaMP8m (PV interneurons), into the PFC of adult C57BL6/N or PV Cre (JAX:008069) mice respectively. To access the PFC, a right-angled microprism attached to a cranial window was implanted into the subdural space within the fissure opposite the site of injection 48 . Methodological details regarding the surgical procedure and training of mice, as well as the in vivo two-photon imaging experiments including image acquisition, quantification and analyses are available in the Supplementary Information . Tissue collection for postmortem analyses The animals were deeply anesthetized with an overdose of pentobarbital (Esconarkon ad us. vet., Streuli Pharma AG, Switzerland) and transcardially perfused with ice-cold artificial cerebrospinal fluid (pH 7.4) 41, 49, 50 . The brains were immediately removed from the skull and either frozen on dry ice and stored at − 80°C until further processing (molecular analyses of KATII expression) or postfixed in 4% PFA for 6 hrs before cryoprotection in 30% sucrose in PBS for 24–48 hours, freezing on dry ice and storage at − 80°C until further processing. Plasma from MIA and CON offspring was collected immediately prior to transcardial perfusion. The atrium was incised, and blood was collected into EDTA-coated blood collection tubes to prevent coagulation. Samples were centrifuged at 2000 × g for 10 minutes to separate plasma, which was then aliquoted and stored at − 20°C until further analysis. Immunohistochemistry and laser-scanning confocal microscopy Immunofluorescent staining and laser-scanning confocal microscopy were used to quantify the intensity of astrocyte markers in the PFC of CON and MIA offspring, the cellular expression pattern of the hM3DGq construct, and c-Fos expression in neuronal subtypes and astrocytes after chemogenetic activation of the latter. Sample processing Fixed brains were cut coronally with a sliding microtome at 30 µm (eight serial sections) and stored at − 20°C in cryoprotectant solution [50 mM sodium phosphate buffer (pH 7.4) containing 15% glucose and 30% ethylene glycol; Sigma-Aldrich, Switzerland] until further processing. Immunofluorescent staining Immunofluorescent stainings were performed according to established protocols 41 , 42 , 49 , 50 . Briefly, the brain sections were incubated with primary antibodies diluted in tris buffer containing 0.2% Triton X-100 and 2% normal serum free-floating under constant agitation (100 rpm) overnight at 4°C. The following day, sections were washed and incubated with secondary antibodies diluted in tris buffer containing 2% normal serum under constant agitation (100 rpm) for 30 min at room temperature. After incubation, which was shielded from light, the sections were washed, mounted onto gelatinized glass slides, coverslipped with Dako fluorescence mounting medium, and stored in the dark at 4°C until image acquisition. Image acquisition Immunofluorescence images were captured by laser scanning confocal microscopy or with Airyscan confocal microscopy (Zeiss LSM800 with Airyscan). To assess the selectivity of hM3DGq construct expression, 6 images randomly selected from 3 consecutive sections within the area of construct expression were acquired per animal using a 40× (oil, NA 1.4) objective with a zoom of 0.45. Higher resolution image stacks for representative images of cell type specific expression of hM3DGq-mCherry were acquired in Airyscan mode using a 40× lens, NA 1.4, oil and processed using the default settings provided by ZEN 2.6 blue edition software (Zeiss, Switzerland). For the c-Fos expression analyses in different cell types of the PFC, 6 randomly selected images within the area of construct expression across 3 consecutive sections were acquired per animal using a 25× (oil, NA 0.8) objective with a zoom of 1. For the intensity analyses of astrocyte markers, 9 images randomly selected from 3 consecutive sections within the PFC were acquired per animal using a 25× (oil, NA 0.8) objective with a zoom of 0.7. Imaging for each experiment were acquired on the same experimental day by an experimenter blinded to the experimental conditions, whereby imaging settings were kept constant throughout an entire imaging day. Image analyses : Image analyses were performed using the ImageJ software by an experimenter blinded to the experimental conditions. Intensity (mean grey value) per astrocyte marker was measured and calculated on z-projected images with a threshold applied to remove background. The mean intensity was than calculated over the 9 images for each animal. For the assessment of selective expression of the hM3DGq construct in astrocytes, the number of mCherry + , S100β + , NeuN + , mCherry + / S100β + , and mCherry + /NeuN + cells was counted within each image. Expression selectivity was then calculated by dividing the number of colocalized cells with the number of total mCherry + cells and multiplied by 100. For the cell-type specific c-Fos mapping within the PFC, the number of c-Fos + cells, the number of target cells, and the number of co-localized cells were counted within each image. The percentage of c-Fos + cells was calculated as follows: (number of co-localized cells/number of target cells) * 100 for each image (referred to as field of view (FOV)). The mean % of c-Fos + cells, the mean number of target cells, and the mean number of c-Fos + cells were then calculated over the 6 images for each animal. Methodological details regarding immunohistochemistry, image acquisition, and analyses are available in the Supplementary Information . Quantification of brain metabolites of the kynurenine pathway Samples of PFC bulk tissue (including anterior cingulate, prelimbic, and infralimbic cortices) were collected and processed as described in the Supplementary Information . Brain metabolites of the KYN pathway were quantified using liquid chromatography-nanoelectrospray ionization tandem mass spectrometry as described in the Supplementary Information . Besides KYN and KYNA, the following metabolites were quantified as well: tryptophan (TRP), quinolinic acid (QUIN), and 3 hydroxykynurenine (3-HK). Quantification of plasma kynurenine and kynurenic acid Plasma samples for KYN and KYNA measurements were diluted (1:2 v/v and 1:10 v/v, respectively) with ultrapure water to a final volume of 100 µl and acidified with 25 µl of 6% perchloric acid. After centrifugation (12,000 × g, 10 min), metabolite levels were measured in 20 µl of the resulting supernatant by high-performance liquid chromatography with fluorimetric detection as described in the Supplementary Information . Quantitative real-time PCR analysis Quantitative RT-PCR was used to measure mAadat RNA levels in PFC extracted from adult C57BL6/N mice expressing no construct (referred to as wild-type) and adult mice that express the KATIIKD-hM3DGq-HA construct in astrocytes. A mouse TaqMan gene expression assay for Aadat (assay ID: Mm00496169_m1, catalogue number: 4331182; Thermo Fisher Scientific, Zurich, Switzerland) was used. The samples were run in 384-well formats in triplicates as multiplexed reactions with the normalizing internal control (36B4). Relative gene expression was calculated with the 2 − ΔΔCt method. All RT-PCRs and analyses were conducted by an experimenter blind to the experimental conditions. Relative changes in mAadat expression were finally compared to the average relative mAadat expression in the PFC of wild-type C57BL6/N mice. Methodological details regarding RNA extraction and TagMan qRT-PCR are available in the Supplementary Information . Western blot analysis Western blot was used to investigate KATII protein levels in total PFC homogenates extracted from adult C57BL6/N male mice expressing no construct (referred to as wild-type) and adult male mice that express KATIIKD-hM3DGq-HA construct in astrocytes. Lysis and sample preparation were performed according to established protocols (see Supplementary Information ). Levels of KAT II (AADAT, rabbit, polyclonal, PA5-88974, Invitrogen,Switzerland, 1:700) were analyzed with reference to Histone 3 (H3, rabbit, monoclonal (D1H2), 4499, Cell Signaling, USA, 1:1000) as the housekeeping control. Methodological details regarding protein separation, transfer and detection are available in the Supplementary Information . Statistical Analyses All statistical analyses were performed using SPSS Statistics (version 29.0, IBM, Armonk, NY, USA) and Prism (version 9.0; GraphPad Software, La Jolla, CA, USA). Statistical significance was set at P < 0.05. Detailed information regarding the statistical analyses used for each experiment is available in the Supplementary Information . Declarations CONTRIBUTIONS V.B., J.F., S.M.S., R.S., E.T., K.D.F., F.H., C.H., A.v.F.C., JC, U.W., and M.W. were involved in the acquisition, analysis, and interpretation of the study data; T.N. and U.M. were involved in the conception and design of the study and analysis and interpretation of the study data; T.N., A.S., S.B., B.W., and U.M. supervised research; T.N., U.M., and A.S. wrote the initial manuscript draft; all authors contributed to the reviewing and editing of the manuscript, and have given final approval for the version to be published. ETHICS DECLARATION All authors declare no competing interests. The funders of the study had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. ACKNOWLEDGMENTS We thank Jean-Charles Paterna and Lazaros Vasilikos from Viral Vector Facility (VVF) of the Neuroscience Center Zurich (ZNZ), Switzerland. We also thank Endre Laczko and Kurt Stefan Schauer from the Functional Genomics Center Zurich (FGCZ), Switzerland, for their technical assistance in liquid chromatography-nanoelectrospray ionization tandem mass spectrometry. This work was financially supported by the Swiss National Science Foundation (grant No. PZ00P3_202149 and grant No. P2ZHP3_174868 awarded to T.N.; grant No. 310030_188524, awarded to U.M.). Additional financial support was provided by the Brain & Behavior Research Foundation (grant No. 30963 awarded to T.N.), the Neuroscience Center Zurich (ZNZ PhD Grant 2022 awarded to T.N), the Olga-Mayenfisch Foundation (awarded to T.N.), and the UZH Candoc Grant, (grant No. FK-25-021 awarded to V.B.). DATA AVAILABILITY All data are available in the main text or the supplementary materials. 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Supplementary Files nrreportingsummary.pdf Reporting summary SupplementaryInformationBeilmannetal.docx Supplementary information SupplementaryTableS2.pdf Supplementary Table S2 Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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08:58:35","extension":"html","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":146200,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-4492882/v1/de9d61d5e7114fbd6e861776.html"},{"id":99215895,"identity":"62dd4df0-1e37-4d5f-ac8b-7e62f9f82bed","added_by":"auto","created_at":"2025-12-30 08:58:35","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":10652808,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAstrocyte stimulation increases KYNA and shifts PFC circuit dynamics. (a)\u003c/strong\u003e Simplified scheme of the AAV. \u003cstrong\u003e(b)\u003c/strong\u003e Expression of the DREADD construct in the target region of interest (medial portion of the PFC) after bilateral stereotaxic injection. Scale bar = 500 μm. \u003cstrong\u003e(c)\u003c/strong\u003e Representative stains confirming cell type selectivity of construct expression. Scale bar = 10 μm. \u003cstrong\u003e(d)\u003c/strong\u003e \u003cem\u003eIn vivo\u003c/em\u003e two-photon Ca\u003csup\u003e2+\u003c/sup\u003e imaging of prefrontal astrocytes in awake mice. Imaging was performed at baseline and after oral administration of either vehicle (VEH) or CNO (1 mg/kg). Ca\u003csup\u003e2+\u003c/sup\u003e responses (means ± SD) were indexed by the F/F0 ratio. \u003cstrong\u003e(e)\u003c/strong\u003e Experimental setup and simplified KYN pathway with relevant metabolites. \u003cstrong\u003e(f)\u003c/strong\u003e Metabolite levels and ratio in the PFC after VEH or CNO treatment. Each data point represents the pooled samples of two mice. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003et\u003c/em\u003e\u003csub\u003e(8)\u003c/sub\u003e = 3.38\u003cstrong\u003e \u003c/strong\u003eand ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, \u003cem\u003et\u003c/em\u003e\u003csub\u003e(8)\u003c/sub\u003e = 6.91.\u003cstrong\u003e (g)\u003c/strong\u003e Schematic illustration of the working hypothesis, in which an astrocyte-induced release of KYNA attenuates parvalbumin (PV) interneuron activity (blue), thereby disinhibiting prefrontal pyramidal cell (PC) activity. \u003cstrong\u003e(h)\u003c/strong\u003e\u003cem\u003e In vivo\u003c/em\u003e two-photon Ca\u003csup\u003e2+\u003c/sup\u003e imaging of prefrontal PV interneurons. Imaging was performed at baseline and after astrocyte stimulation with CNO (1 mg/kg). Ca\u003csup\u003e2+\u003c/sup\u003e responses were indexed by the F/F0 ratio. \u0026nbsp;\u003cstrong\u003e(i)\u003c/strong\u003e Schematic illustration of cell-type-specific c-Fos mapping after astrocyte stimulation. \u003cstrong\u003e(j)\u003c/strong\u003e PV (red) and c-Fos (green) expression in the PFC after VEH or CNO treatment; arrowhead denotes a c-Fos-positive PV interneuron. The bar plot depicts the % of c-Fos positive PV cells. ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, \u003cem\u003et\u003c/em\u003e\u003csub\u003e(14)\u003c/sub\u003e = 4.19. \u003cstrong\u003e(k)\u003c/strong\u003e CaMKII (red) and c-Fos (green) expression in the PFC after VEH or CNO treatment; arrowheads denote c-Fos positive pyramidal cells. The bar plot depicts the % of c-Fos positive CaMKII pyramidal cells after VEH or CNO treatment. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003et\u003c/em\u003e\u003csub\u003e(14)\u003c/sub\u003e = 2.23. Scale bars = 15 μm. Data are means ± SEM with individual values overlaid.\u003c/p\u003e","description":"","filename":"Fig1Beilmannetal2025.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4492882/v1/633ecd22c240d5bb3ec59e7c.jpg"},{"id":99215891,"identity":"d46eb32c-0e1f-4b3f-945f-5dc9f09bf8b2","added_by":"auto","created_at":"2025-12-30 08:58:35","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1524912,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAstrocyte stimulation in PFC impairs cognition and pre-attentive filtering (a)\u003c/strong\u003e Scheme of experimental setup. \u003cstrong\u003e(b)\u003c/strong\u003e Absolute exploration times of the temporally remote and recent objects (line plots) and temporal order memory index (bar plot) in the temporal order memory test for objects. ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, \u003cem\u003et\u003c/em\u003e\u003csub\u003e(18)\u003c/sub\u003e = 4.01.\u003cstrong\u003e (c)\u003c/strong\u003e Percent spontaneous alternation and number of arm entries in the Y-maze test of working memory. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01,\u003cem\u003e t\u003c/em\u003e\u003csub\u003e(18)\u003c/sub\u003e = 3.61.\u003cstrong\u003e (d) \u003c/strong\u003ePrepulse inhibition (PPI) test of pre-attentive filtering. The line plots show % PPI as a function of prepulse intensity (71, 77 and 83 dB\u003csub\u003eA\u003c/sub\u003e) for each of the three pulse conditions (P100, P110 and P120, which correspond to pulse intensities of 100, 110 and 120 dB\u003csub\u003eA\u003c/sub\u003e). The bar plot depicts the mean % PPI across all prepulse and pulse intensities. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(1,18)\u003c/sub\u003e = 9.98. All data are means ± SEM with individual values overlaid.\u003c/p\u003e","description":"","filename":"Fig2Beilmannetal2025.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4492882/v1/06807789f467950761c80cd6.jpg"},{"id":99215892,"identity":"0ebd4d4a-c274-4902-8d37-c7ed843a31cf","added_by":"auto","created_at":"2025-12-30 08:58:35","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2204008,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of KYNA synthesis reverses cognitive impairments and normalizes PFC cortical dynamics. (a)\u003c/strong\u003e Schematic representation of the pharmacological rescue study. hM3DGq-expressing mice were pretreated with the KAT II inhibitor PF-04859989 (0, 1, or 10 mg/kg, i.p.) 2.5 hours before receiving CNO or VEH, and 3 hours before they were subjected to behavioral and cognitive testing. \u003cstrong\u003e(b)\u003c/strong\u003e Temporal order memory test for objects. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, based on post-hoc test following ANOVA (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e(3,36)\u003c/sub\u003e = 11.96, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). \u003cstrong\u003e(c)\u003c/strong\u003e Y-maze test of working memory. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 and **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, based on post-hoc test following ANOVA (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e(3,36)\u003c/sub\u003e = 5.60, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). \u003cstrong\u003e(d)\u003c/strong\u003e Prepulse inhibition test. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 and **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, based on post-hoc test following ANOVA (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e(3,36)\u003c/sub\u003e = 4.46, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). \u003cstrong\u003e(e) \u003c/strong\u003eTemporal order memory test in hM3DGq-expressing mice pretreated with 0 or 10 mg/kg PF-04859989 2.5 hours before receiving CNO or VEH. ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 and ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, based on post-hoc test following one-way ANOVA (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e(2,21)\u003c/sub\u003e = 19.43, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001). 2 hours after cognitive testing tissue was collected for cell-type-specific c-Fos mapping. \u003cstrong\u003e(f)\u003c/strong\u003e % of c-Fos-positive PV interneurons. ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, based on post-hoc test following one-way ANOVA (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e(2,21)\u003c/sub\u003e = 11.04, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). \u003cstrong\u003e(g)\u003c/strong\u003e % of c-Fos-positive CaMKII pyramidal cells. ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, based on post-hoc test following one-way ANOVA (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e(2,21)\u003c/sub\u003e = 54.56, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001). All data are means ± SEM with individual values overlaid.\u003c/p\u003e","description":"","filename":"Fig3Beilmannetal2025.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4492882/v1/58bf328e69a4cbd45e50163a.jpg"},{"id":99317160,"identity":"1d65475e-e727-4253-b747-e75f7dbc7ab9","added_by":"auto","created_at":"2025-12-31 16:29:43","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6550190,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKAT II knockdown prevents astrocyte-mediated cognitive and neuronal deficits. (a)\u003c/strong\u003e Simplified scheme of the AAV. \u003cstrong\u003e(b)\u003c/strong\u003e \u003cem\u003eAadat\u003c/em\u003e mRNA expression in bulk PFC tissue from KATII\u003csup\u003eKD\u003c/sup\u003e or WT mice. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003et\u003c/em\u003e\u003csub\u003e(14)\u003c/sub\u003e = 3.449. \u003cstrong\u003e(c)\u003c/strong\u003e Representative KAT II Western blot with PFC lysates from WT (W) and KATII\u003csup\u003eKD\u003c/sup\u003e (K) mice. Protein ladder (L) and histone 3 (H3) as loading control. Arrows indicate KAT II monomer and homodimer bands. KAT II monomer (left) and KAT II dimer (right) protein levels. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003et\u003c/em\u003e\u003csub\u003e(14)\u003c/sub\u003e = 2.286; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003et\u003c/em\u003e\u003csub\u003e(14)\u003c/sub\u003e = 3.594. \u003cstrong\u003e(d)\u003c/strong\u003e Representative stains confirming cell type selectivity of construct expression. Scale bar = 20 μm. \u003cstrong\u003e(e)\u003c/strong\u003e \u003cem\u003eIn vivo\u003c/em\u003e two-photon Ca\u003csup\u003e2+\u003c/sup\u003e imaging of prefrontal astrocytes in anesthetized animals. Ca\u003csup\u003e2+\u003c/sup\u003e responses (F/F0 ratio, means ± SD) were measured at baseline and after VEH and CNO (1 mg/kg) treatment. \u003cstrong\u003e(f)\u003c/strong\u003e Scheme of experimental setup.\u003cstrong\u003e (g) \u003c/strong\u003eTemporal order memory test for objects.\u003cstrong\u003e \u003c/strong\u003e*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, based on post-hoc test following two-way ANOVA with a significant interaction (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e(1,35)\u003c/sub\u003e = 8.22, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). \u003cstrong\u003e(h)\u003c/strong\u003e Y-maze test of working memory.\u003cstrong\u003e \u003c/strong\u003e*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, based on post-hoc test following two-way ANOVA with a significant interaction (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e(1,35)\u003c/sub\u003e = 8.22, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). \u003cstrong\u003e(i) \u003c/strong\u003ePrepulse inhibition test. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, based on post-hoc test following two-way ANOVA with a significant interaction (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e(1,35)\u003c/sub\u003e = 4.18, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). \u003cstrong\u003e(j) \u003c/strong\u003eAbsolute exploration times of the temporally remote and recent objects (line plots) and temporal order memory index (bar plot) in the temporal order memory test for objects.\u003cstrong\u003e (k)\u003c/strong\u003e Quantification of c-Fos-positive PV+ cells after VEH or CNO treatment. \u003cstrong\u003e(l)\u003c/strong\u003e Quantification of c-Fos positive CaMKII pyramidal cells after VEH or CNO treatment. Scale bars = 15 μm. All data are means ± SEM with individual values overlaid.\u003c/p\u003e","description":"","filename":"Fig4Beilmannetal2025.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4492882/v1/020012b7b02f7223827f7167.jpg"},{"id":99215899,"identity":"2fabec36-c550-4cf7-9114-b40ff80d09e4","added_by":"auto","created_at":"2025-12-30 08:58:35","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4193053,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibiting KYNA synthesis reverses MIA-induced cognitive deficits. (a)\u003c/strong\u003e Pregnant mice were exposed to MIA, induced by poly(I:C) administration, or vehicle (CON) on gestation day (GD) 12. The resulting male offspring underwent behavioral and cognitive testing in adulthood (postnatal week 12 (12 WKS)). \u003cstrong\u003e(b)\u003c/strong\u003e Absolute exploration time of the temporally remote and recent objects (line plots) and temporal order memory index (bar plot) in the temporal order memory test for objects. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003et\u003c/em\u003e\u003csub\u003e(28)\u003c/sub\u003e = 2.36. \u003cstrong\u003e(c)\u003c/strong\u003e Representative stains against GFAP in the PFC and corresponding mean intensity (mean grey value (MGV)) measured in the PFC. ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, \u003cem\u003et\u003c/em\u003e\u003csub\u003e(28)\u003c/sub\u003e = 3.77.\u003cstrong\u003e (d) \u003c/strong\u003ePearson’s product moment correlations between prefrontal GFAP intensity and temporal order memory index. \u003cstrong\u003e(e)\u003c/strong\u003e Simplified scheme of the peripheral KYN pathway depicting blood-brain-barrier (BBB) permeability of KYN, serving as the precursor of the downstream neuroactive metabolite KYNA, (produced in astrocytes, as depicted in blue) and quinolinic acid. Plasma levels of KYN and KYNA. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003et\u003c/em\u003e\u003csub\u003e(28)\u003c/sub\u003e = 2.98. \u003cstrong\u003e(f)\u003c/strong\u003e Pearson’s product moment correlations between plasma KYN levels and temporal order memory index. \u003cstrong\u003e(g)\u003c/strong\u003e Pearson’s product moment correlations between plasma KYN levels and prefrontal GFAP intensity. Scale bar = 20 μm. All data except correlations are means ± SEM with individual values overlaid. \u003cstrong\u003e(h)\u003c/strong\u003e Schematic representation of the pharmacological rescue study. \u003cstrong\u003e(i)\u003c/strong\u003e Temporal order memory index for CON or MIA offspring receiving 0 or 10 mg/kg PF-04859989. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, based on post-hoc test following ANOVA (main effect of prenatal treatment: \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(1,40)\u003c/sub\u003e = 4.38, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; 2-way interaction:\u0026nbsp; \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(1,40)\u003c/sub\u003e = 4.57, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). All data are means ± SEM with individual values overlaid.\u003c/p\u003e","description":"","filename":"Fig5Beilmannetal2025.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4492882/v1/725caaff998706f1f370486e.jpg"},{"id":99323976,"identity":"c79e1f1a-401b-42a7-8d22-add4229c1910","added_by":"auto","created_at":"2025-12-31 16:46:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":26746739,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4492882/v1/d408aacc-d415-42e4-8648-7f34cb468e4b.pdf"},{"id":99317736,"identity":"9ccb767b-06f3-43f6-a7d3-b0d906ee2444","added_by":"auto","created_at":"2025-12-31 16:30:39","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1666905,"visible":true,"origin":"","legend":"Reporting summary","description":"","filename":"nrreportingsummary.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4492882/v1/303e6a1f3cde1b05893a22ac.pdf"},{"id":99215905,"identity":"ee20ffea-24e4-4bf6-8042-9ee1e1ce3ebe","added_by":"auto","created_at":"2025-12-30 08:58:35","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":13095340,"visible":true,"origin":"","legend":"Supplementary information","description":"","filename":"SupplementaryInformationBeilmannetal.docx","url":"https://assets-eu.researchsquare.com/files/rs-4492882/v1/32d6ffcecbb1c680227068be.docx"},{"id":99215893,"identity":"2e5711ae-9cd9-443f-91ae-6ab554a6e32e","added_by":"auto","created_at":"2025-12-30 08:58:35","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":454774,"visible":true,"origin":"","legend":"Supplementary Table S2","description":"","filename":"SupplementaryTableS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4492882/v1/b7d186df995f71797eb28706.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Astroglial disinhibition of cortical circuits disrupts cognition via kynurenic acid","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAstrocytes are increasingly recognized as active participants in neural circuit dynamics. In addition to their classical roles in metabolic and homeostatic support, they modulate synaptic transmission, shape network activity, and influence behavioral states\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Astrocyte physiology is markedly altered in many neurological and psychiatric conditions\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Inflammatory and disease-associated environmental stimuli trigger changes in astrocytic gene expression, intracellular signaling, and metabolic output. These reactive states have been observed in disorders ranging from epilepsy and neurodegeneration to schizophrenia and depression\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite growing recognition that astrocytes are affected in central nervous system (CNS) disorders, the consequences of these altered states for circuit function and cognition remain poorly understood. This question is especially pertinent to the prefrontal cortex (PFC), a region central to higher-order cognitive function, where circuit dysregulation is strongly linked to cognitive deficits in psychiatric disorders\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Cortical circuits in the PFC depend on the precise balance of excitatory and inhibitory activity to sustain working memory, attention, and sensorimotor integration\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Maintenance of this balance depends on the coordinated interaction between inhibitory interneurons and pyramidal cells, with fast-spiking parvalbumin-positive (PV+) interneurons and their NMDAR activity playing a key role\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. However, whether astrocytes influence these circuits, and the molecular mechanisms by which altered astrocyte activity impacts prefrontal computation and behavior, are not well defined.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAstrocyte stimulation in the PFC elevates KYNA and alters cortical circuit activity\u003c/h2\u003e \u003cp\u003eTo examine how altered astrocyte states impact PFC circuit function, we selectively increased astrocyte activity using chemogenetics. To this end, Gq-signaling was activated in PFC astrocytes of adult mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-c). \u003cem\u003eIn vivo\u003c/em\u003e two-photon Ca\u0026sup2;⁺ imaging in awake mice confirmed robust and repeatable clozapine-\u003cem\u003eN\u003c/em\u003e-oxide (CNO)-induced Ca\u0026sup2;⁺ elevation in transduced astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, \u003cb\u003eSupplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Using this approach, we then examined whether altering PFC astrocyte states shifts the metabolic pathway of kynurenine (KYN) degradation. We focused on this pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee) because its downstream metabolite, kynurenic acid (KYNA), is an endogenous NMDA receptor antagonist implicated in cognitive dysfunction and predominantly synthesized by astrocytes \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Notably, PFC KYNA levels and the KYNA/KYN ratio were significantly elevated in CNO-treated mice, while KYN levels and other pathway metabolites remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef, \u003cb\u003eSupplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eAccording to the excitatory-inhibitory (E/I) imbalance hypothesis, NMDA receptor antagonists, such as ketamine, disrupt prefrontal circuit function by suppressing parvalbumin-positive (PV+) interneuron activity, leading to disinhibition of pyramidal neurons and cognitive impairment \u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Since KYNA is an endogenous NMDAR antagonist primarily produced by astrocytes \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, we hypothesized that astrocyte-induced elevated KYNA may similarly reduce PV\u0026thinsp;+\u0026thinsp;interneuron activity and thereby shift prefrontal circuit dynamics toward excitation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). To test this, we combined \u003cem\u003ein vivo\u003c/em\u003e two-photon Ca\u003csup\u003e2+\u003c/sup\u003e imaging in PV\u0026thinsp;+\u0026thinsp;interneurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh) with cell-type-specific c-Fos mapping (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei) following chemogenetic activation of prefrontal astrocytes. In line with our hypothesis, CNO treatment reduced PV\u0026thinsp;+\u0026thinsp;interneuron activity as evident by the reduction in spike activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh) and c-Fos expression in PV\u0026thinsp;+\u0026thinsp;interneurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej, \u003cb\u003eSupplementary Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003ea\u003c/b\u003e), while increasing c-Fos in CaMKII\u0026thinsp;+\u0026thinsp;excitatory pyramidal neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ek, \u003cb\u003eSupplementary Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eb\u003c/b\u003e). No changes were observed in somatostatin-positive (SST+) interneurons (\u003cb\u003eSupplementary Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003ec\u003c/b\u003e), indicating a selective effect on PV\u0026thinsp;+\u0026thinsp;cells. Together, these findings demonstrate that altering PFC astrocyte states increases KYNA and suppresses PV\u0026thinsp;+\u0026thinsp;interneuron activity, leading to disinhibition of pyramidal neurons. This shift in circuit dynamics could provide the mechanistic basis through which astrocytes could contribute to cognitive impairment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAstrocyte stimulation impairs memory and pre-attentive filtering\u003c/h3\u003e\n\u003cp\u003eTo assess whether astrocyte-induced changes in KYNA and circuit dynamic in the PFC translate into cognitive dysfunctions, we subjected mice to different behavioral and cognitive tests following CNO administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Chemogenetic activation of prefrontal astrocytes impaired performance across several PFC-dependent cognitive domains. In the temporal order memory test for episodic-like memory, CNO-treated male mice failed to discriminate object recency compared to vehicle (VEH) treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In the Y-maze test for working memory, astrocyte stimulation reduced spontaneous alternation without affecting total entries (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). These effects occurred without changes in anxiety-like behavior or locomotion (\u003cb\u003eSupplementary Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/b\u003e), demonstrating selective effects of astrocyte activation on cognitive performance. Furthermore, CNO-treatment led to the disruption of prepulse inhibition (PPI) of the acoustic startle reflex (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), a form of pre-attentive filtering deficient in various psychiatric disorders\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e and impaired by elevated endogenous brain KYNA\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Control mice expressing a GFAP-driven EGFP construct showed no behavioral effects of CNO (\u003cb\u003eSupplementary Fig. S5\u003c/b\u003e), ruling out off-target drug effects. Similar deficits were observed in female hM3DGq mice (\u003cb\u003eSupplementary Fig. S6\u003c/b\u003e), showing that the astrocyte-induced cognitive disruption is present in male and female mice. Together, these findings demonstrate that selective stimulation of prefrontal astrocytes impairs cognitive functions and pre-attentive filtering. The shift towards increased KYNA may underlie the observed changes in circuit activity and behavior, warranting further testing of causality.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eKYNA mediates astrocyte-induced cognitive deficits via PV interneuron suppression\u003c/h3\u003e\n\u003cp\u003eTo test whether elevated KYNA causally mediates the cognitive impairments and circuit alterations, we first pharmacologically inhibited its synthesis. We used PF-04859989, a brain-penetrant inhibitor of KAT II, which efficiently lowers brain KYNA levels after systemic administration\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Mice received PF-04859989 (1 mg/kg or 10 mg/kg) 2.5 hours before CNO-induced astrocyte activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). While sub-threshold dose (1 mg/kg) had no effect, the higher dose (10 mg/kg) fully restored KYNA levels, temporal order memory, working memory, and PPI in CNO-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, \u003cb\u003eSupplementary Fig. S7a\u003c/b\u003e). These effects were not due to general cognitive enhancement. When the same doses of PF-04859989 were administered to non-activated control mice, no significant changes in cognitive performance were observed (\u003cb\u003eSupplementary Fig. S7b-e\u003c/b\u003e). Thus, KAT II inhibition selectively reversed the cognitive deficits induced by astrocyte activation, without baseline pro-cognitive effects. We next examined whether KYNA mediates the shift in cortical circuit dynamics. Administration of 10 mg/kg PF-04859989 prior to CNO treatment restored temporal order memory performance (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee) and normalized c-Fos levels in both PV\u0026thinsp;+\u0026thinsp;interneurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, \u003cb\u003eSupplementary Fig. S8a\u003c/b\u003e) and CaMKII\u003csup\u003e+\u003c/sup\u003e pyramidal neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg, \u003cb\u003eSupplementary Fig. S8b\u003c/b\u003e), without affecting SST\u003csup\u003e+\u003c/sup\u003e interneurons (\u003cb\u003eSupplementary Fig. S8c\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further validate the role of astrocyte-derived KYNA in driving these dysfunctions, we next used a genetic approach to block KYNA synthesis selectively in activated prefrontal astrocytes. We hereby injected custom-made AAVs expressing four unique miRNA-adapted shRNAs targeting \u003cem\u003emAadat\u003c/em\u003e (referred to as KATII\u003csup\u003eKD\u003c/sup\u003e) along with hM3DGq and an HA tag into the PFC of male mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). This design enabled simultaneous chemogenetic activation and selective knockdown of KAT II in prefrontal astrocytes. Knockdown efficacy was confirmed by reduced \u003cem\u003emAadat\u003c/em\u003e mRNA and decreased protein levels of both the ~\u0026thinsp;47 kDa KAT II monomer and its active\u0026thinsp;~\u0026thinsp;94 kDa homodimer \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e in PFC homogenates of KATII\u003csup\u003eKD\u003c/sup\u003e mice compared to wild-type controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, c). Immunohistochemistry confirmed astrocyte-specific expression in the PFC, as observed with the original hM3DGq-mCherry construct (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). \u003cem\u003eIn vivo\u003c/em\u003e two-photon Ca\u0026sup2;⁺ imaging in anesthetized mice demonstrated that hM3DGq-mediated astrocyte activation remained functional in the KATII\u003csup\u003eKD\u003c/sup\u003e condition, as indicated by CNO-induced Ca\u0026sup2;⁺ elevations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eWe next assessed whether reducing KAT II expression was sufficient to prevent astrocyte-induced cognitive dysfunction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). KAT II knockdown prevented the emergence of episodic-like memory (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg) and working memory deficits (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh), as well as the disruption of PPI of the acoustic startle reflex (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei). A concurrent normalization was observed on the level of PV\u0026thinsp;+\u0026thinsp;interneurons and pyramidal cell activity. Mice expressing KATII\u003csup\u003eKD\u003c/sup\u003e-hM3DGq-HA that failed to show any deficits in temporal order memory (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej), working memory (\u003cb\u003eSupplementary Fig. S9a\u003c/b\u003e), or PPI (\u003cb\u003eSupplementary Fig. S9b\u003c/b\u003e) upon CNO treatment, also displayed unchanged levels of c-Fos expression in both PV\u0026thinsp;+\u0026thinsp;and CaMKII\u0026thinsp;+\u0026thinsp;neurons compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek, l; \u003cb\u003eSupplementary Fig. S9c\u003c/b\u003e). These findings confirm that circuit alterations and cognitive impairments were dependent on astrocyte-derived KYNA. Together, these findings identify an astrocyte-KYNA-interneuron axis, whereby astrocyte-derived KYNA impair cognition via impeding PFC E/I balance by selectively suppressing PV\u0026thinsp;+\u0026thinsp;interneuron activity, leading to disinhibition of pyramidal neurons.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eKYNA contributes to cognitive dysfunction in a neuropsychiatric risk model\u003c/h3\u003e\n\u003cp\u003eTo examine how altered astrocyte states and kynurenine metabolism relate to cognitive dysfunction within a translationally relevant disease framework, we used a mouse model of maternal immune activation (MIA), an established environmental risk factor for neurodevelopmental disorders with cognitive impairments \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. MIA was induced by prenatal administration of poly(I:C), a viral mimetic that triggers an acute inflammatory response during fetal development (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Consistent with previous findings\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, MIA impaired the ability of offspring to discriminate objects based on the temporal presentation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), which emerged in the absence of concomitant changes in basal locomotor and innate anxiety-like behavior (\u003cb\u003eSupplementary Fig. S10\u003c/b\u003e). We next assessed whether MIA alters astrocyte states in the PFC. GFAP immunoreactivity was significantly elevated in MIA offspring, consistent with increased astrocyte reactivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). While other astrocytic markers remained unchanged (\u003cb\u003eSupplementary Fig. S11\u003c/b\u003e), individual GFAP levels negatively correlated with cognitive performance in MIA but not control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed), linking astrocyte reactivity to cognitive impairment.\u003c/p\u003e \u003cp\u003eWe then investigated whether these behavioral and glial changes were accompanied by alterations in kynurenine (KYN) metabolism. Compared to controls, MIA offspring displayed elevated plasma KYN levels, the blood brain barrier (BBB)-permeable and immediate precursor of central KYNA\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, without changes in peripheral BBB-impermeable KYNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Notably, KYN levels negatively correlated with memory performance (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef) and positively with prefrontal GFAP intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg), suggesting a coordinated relationship between systemic KYN, astrocyte reactivity, and behavioral deficits.\u003c/p\u003e \u003cp\u003eTo test whether elevated KYNA contributes causally to cognitive impairments, adult MIA and control offspring were treated with PF-04859989 (10 mg/kg) or vehicle before testing (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh). In MIA offspring, PF-04859989 restored temporal order memory to control levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei). This effect was not attributable to a general enhancement of cognitive performance, as PF-04859989 treatment had no impact in control offspring, indicating specificity to the pathophysiological state. Together, these findings link heightened prefrontal astrocyte reactivity and KYNA to impaired episodic-like memory performance in a mouse model of psychiatric disease risk, supporting an astrocyte-KYNA contribution to cognitive dysfunction under pathophysiological conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOur study reveals a previously unrecognized astrocyte-interneuron signaling mechanism that shapes prefrontal circuit function and cognition. We demonstrate that increased astrocyte activity impairs cognitive performance by elevating the neuroactive metabolite KYNA, the brain\u0026rsquo;s only known endogenous NMDAR antagonist\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, which selectively suppresses PV\u0026thinsp;+\u0026thinsp;interneuron activity and in turn disinhibits pyramidal neurons. The KYNA-driven excitatory-inhibitory imbalance and cognitive deficits are reminiscent of those induced by synthetic NMDAR antagonists such ketamine\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, PCP\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, or MK-801\u003csup\u003e25\u003c/sup\u003e. While prior work has implicated astrocyte-derived KYNA in the modulation of excitatory transmission\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, our study establishes the first causal link between astrocyte-driven KYNA elevation, cell-type-specific neuronal effects, and cognitive impairment in vivo.\u003c/p\u003e \u003cp\u003eOur findings complement recent work on astrocyte-mediated state transitions\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e by identifying a defined metabolic signaling axis (astrocyte-KYNA-interneuron) that disrupts local circuit balance and cognitive performance. Importantly, this axis engages PV\u0026thinsp;+\u0026thinsp;interneurons, a neuronal population consistently implicated in psychiatric disease pathophysiology\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, and positions astrocytes as upstream modulators of their activity via KYNA. Concurrent to this mechanistic link, our data reinforce the relevance of the KYN pathway in psychiatric diseases. Elevation of prefrontal KYNA levels in the astrocyte DREADD model parallels findings in patients with schizophrenia or bipolar disorder, where increased KYNA has been detected in cortical or cerebrospinal fluid samples\u003csup\u003e\u003cspan additionalcitationids=\"CR31 CR32 CR33\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Although the exact sources of KYNA in human disease remain unclear, our results suggest that astrocytes are not only capable of producing KYNA under pathophysiological conditions, but also directly contribute to cognitive dysfunction when KYNA levels are elevated.\u003c/p\u003e \u003cp\u003eWe also demonstrate that astrocyte reactivity and KYN metabolism correlate with cognitive deficits in offspring subjected to MIA, a well-established neurodevelopmental model of psychiatric disease risk \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. While MIA induces widespread changes in brain development and processes \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, our data point to a prefrontal astrocyte-KYNA axis as a disease-relevant contributor to cognitive impairments. This is consistent with human studies linking elevated peripheral KYN levels to cognitive deficits in a subset of schizophrenia patients\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Moreover, pharmacological inhibition of KAT II, the enzyme converting KYN into KYNA, rescued memory and sensorimotor gating deficits in both MIA and DREADD models, without inducing pro-cognitive effects in control animals. These findings identify KAT II as a potential therapeutic target for conditions marked by glial dysregulation and cognitive dysfunction.\u003c/p\u003e \u003cp\u003eWhile our study focused specifically on astrocyte activity in the PFC, the astrocyte-KYNA-interneuron signaling axis we define may operate in other brain regions and disease contexts as well. The ability of astrocytes to reshape local circuit dynamics via metabolic modulation of interneuron activity expands our understanding of glial contributions to behavior and identifies a mechanistic link between astrocyte reactivity, KYN metabolism, and cognitive dysfunction. Given the prominence of astrocyte dysregulation, elevated KYNA levels, and PV interneuron deficits across psychiatric disorders such as schizophrenia and bipolar disorder\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, these findings provide a conceptual and mechanistic framework for targeting astrocyte-derived metabolites in the treatment of cognitive symptoms.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eAll experiments were performed using male or female C57BL6/N mice (Charles Rivers, Sulzfeld, Germany). They were group-housed (4\u0026ndash;5 animals per cage) in individually ventilated cages under a reversed light\u0026ndash;dark cycle. All animals had \u003cem\u003ead libitum\u003c/em\u003e access to standard rodent chow and water throughout the entire study. All procedures were conducted during the dark cycle and had been previously approved by the Cantonal Veterinarian\u0026rsquo;s Office of Zurich. All efforts were made to minimize the number of animals used and their suffering. Details about housing conditions and an overview of the different cohorts of animals and the respective numbers used are provided in the \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDREADD system\u003c/h3\u003e\n\u003cp\u003eThe DREADD system was based on a recombinant adeno-associated virus serotype 9 (AAV9) that expresses hM3DGq under the astrocyte-specific promoter hgfaABC1D (hGFAP), encompassing a fluorescent mCherry tag (AAV9-hGFAP-hM3DGq-mCherry; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Some experiments involved an EGFP-tagged control virus (ConV) with the same promoter (AAV9-hGFAP-EGFP) and the GCaMP6s Ca\u003csup\u003e2+\u003c/sup\u003e sensor (AAV9-hGFAP-GCaMP6s). In addition, some experiments involved a custom-made virus that expresses 4 unique miRNA-adapted shRNA hairpins directed against \u003cem\u003emAadat\u003c/em\u003e mRNA together with hM3DGq under the same hGFAP promoter (AAV9-hGFAP-chI[4x:sh(mAadat)]-HA-hM3DGq, herein referred to as AAV9-hGFAP-KATIIKD-hM3DGq-HA), see below and \u003cb\u003eSupplementary Information\u003c/b\u003e). All AAVs were produced and purchased from the Viral Vector Facility of the University of Zurich, Switzerland (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.vvf.uzh.ch\" target=\"_blank\"\u003ewww.vvf.uzh.ch\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.vvf.uzh.ch\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and were injected into the PFC using bilateral stereotaxic injections (stereotaxic coordinates: anteroposterior [AP]\u0026thinsp;=\u0026thinsp;+\u0026thinsp;1.8 mm, mediolateral [ML]\u0026thinsp;=\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 mm, dorsoventral [DV]\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;1.9 mm) as described in the \u003cb\u003eSupplementary Information\u003c/b\u003e. hM3DGq was activated with 1 mg/kg clozapine-N-oxide (CNO, BML-NS105-0025, Enzo Life Sciences, Switzerland) dissolvedac in 0.9% NaCl (B. Braun, Switzerland). The dose of 1 mg/kg was chosen based on previous chemogenetic studies in rodents\u003csup\u003e\u003cspan additionalcitationids=\"CR40 CR41\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Treatment with vehicle (VEH; 0.9% NaCl) served as control treatment. ConV-expressing mice receiving CNO or VEH were used to exclude non-selective effects of CNO\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCNO (1 mg/kg) or VEH were given via the micropipette-guided drug administration (MDA) method, a non-invasive oral administration technique described in detail elsewhere\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Experiments involving \u003cem\u003ein vivo\u003c/em\u003e two-photon imaging in anesthetized mice VEH or CNO (1mg/kg) were injected s.c. with an injection volume of 2 mL/kg (\u003cb\u003eSupplementary Information\u003c/b\u003e).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eKynurenine aminotransferase II inhibition\u003c/h2\u003e \u003cp\u003eKynurenine aminotransferase II (KAT II) was inhibited with 1 mg/kg or 10 mg/kg PF-04859989 hydrochloride (PZ0250, Sigma-Aldrich), which was dissolved in sterile water and freshly prepared prior to each experiment. For animals subjected to behavioral testing, 1 or 10 mg/kg PF-04859989, or sterile water (vehicle, 0 mg/kg) only, was injected i.p. using an injection volume of 5 mL/kg 3 hrs prior to each behavioral test. For postmortem analyses of c-Fos expression, sterile water (vehicle) or 10 mg/kg PF-04859989 was administered either 5 hours (behaviorally na\u0026iuml;ve animals) or 8 hrs (behaviorally tested animals) prior to tissue collection. The doses and post-injection interval were chosen based on previous dose-response studies in rodents\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAstrocyte selective knockdown of Kynurenine aminotransferase II inhibition.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo selectively knockdown KAT II expression in astrocytes, we employed the miRNA-adapted short/small hairpin (sh) RNA (shRNAmir) strategy\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Construct design and AAV production were conducted by the Viral Vector Facility, University of Zurich, Switzerland according to established protocols. shRNA silencing constructs were custom designed against the \u003cem\u003emAadat\u003c/em\u003e transcript NM_011834.2. Four 21-mer shRNA sequences were selected based on their target selectivity and highest probability ranking for knockdown efficacy. The shRNA sequences were: 5\u0026rsquo;-GCAACAACCCTACAGGCAACT-3\u0026rsquo;, 5\u0026rsquo;-GGTTGAGAGTAGGGTTTATGA-3\u0026rsquo;, 5\u0026rsquo;-GGTTTATGACTGGCCCTAAGA-3\u0026rsquo;, and 5\u0026rsquo;-GGGTTTCCTGGCTCATATTGA-3\u0026rsquo;. An shRNAmir-E cassette containing the four shRNAs was cloned into a pssAAV-2-hGFAP-HA_hM3D(Gq)-bGHp(A) backbone, to generate the pssAAV-2-hGFAP-chI[4x:sh(mAadat)]-HA-hM3DGq-bGHp(A) plasmid. The construct was then packaged in AAV9 to produce the AAV9-hGFAP-chI[4x:sh(mAadat)]-HA-hM3DGq (referred to as AAV9-hGFAP-KATIIKD-hM3DGq-HA or short KATIIKD-hM3DGq), allowing for region and cell type-selective knockdown of KAT II expression.\u003c/p\u003e \u003cp\u003eKnockdown efficacy was assessed 4 weeks after construct expression by real-time quantitative PCR (RT-qPCR) and Western blot analyses using total RNA and proteins extracted from PFC of male C57BL6/N mice expressing the KATIIKD-hM3DGq construct in prefrontal astrocytes or C57BL6/N control male mice expressing no construct (referred to as wild-type). Methodological details regarding RT-qPCR and Western blot experiments are available in the \u003cb\u003eSupplementary Information\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMaternal immune activation model\u003c/h2\u003e \u003cp\u003eTwo independent cohorts of time-pregnant mice were used in this study. The cohorts were generated via on-site breeding under identical experimental conditions as described in detail in the \u003cb\u003eSupplementary Information\u003c/b\u003e. Cohort 1 was used for the assessment of cognitive deficits, followed by post-mortem analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Cohort 2 was used for the pharmacological rescue study of the cognitive deficits using the KATII inhibitor PF-04859989 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). On gestational day (GD) 12, pregnant mice were randomly assigned to a single injection of poly(I:C) (potassium salt, P9582, Sigma\u0026ndash;Aldrich, Buchs, St. Gallen, Switzerland) or treatment with endotoxin-free 0.9% NaCl (B. Braun, Melsungen, Switzerland) vehicle solution. Male offspring to CON and MIA dams were weaned and housed in groups of 2 to 5 per cage as described above. Behavioral testing in both cohorts commenced when the offspring reached 12 weeks of age. A minimum of 1-week resting period was imposed after behavioral testing before the animals were killed and tissue was collected for subsequent post-mortem analyses (see below).\u003c/p\u003e \u003cp\u003eMethodological details regarding the MIA model, including timed-mating, treatment, birth conditions and weaning of offspring, can be obtained in the \u003cb\u003eSupplementary Information\u003c/b\u003e and the reporting guideline checklist for the MIA model\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e provided in \u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBehavioral and cognitive testing\u003c/h2\u003e \u003cp\u003eBehavioral and cognitive testing included temporal order memory test for objects, a spontaneous alternation task for working memory in the Y-maze, a prepulse inhibition (PPI) test of the acoustic startle reflex for pre-attentive filtering, as well as tests assessing locomotor activity and innate anxiety-like behavior (open field and light-dark box tests). Mice undergoing multiple behavioral tests were given a minimum inter-test interval (ITI) of 48 hours between tests with the exception between open field test and the temporal order memory test. Because the open field test also serves as habituation to the arena for the temporal order memory test, a 24-hour ITI was implemented between the two tests. For the hM3DGq-based DREADD model, 30 min prior to each behavioral test mice were treated once with VEH or CNO (1mg/kg) via MDA. A detailed description of the methodological procedures and rationale of inclusion for each behavioral and cognitive test are provided in the \u003cb\u003eSupplementary Information\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003etwo-photon Ca\u003c/b\u003e\u003csup\u003e\u003cb\u003e2+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eimaging of prefrontal astrocytes in mice using microprisms\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vivo\u003c/em\u003e Ca\u003csup\u003e2+\u003c/sup\u003e imaging using two-photon microscopy in awake or anesthetized mice was applied to ascertain the effectiveness and temporal dynamics of hM3DGq-based elevation of Ca\u003csup\u003e2+\u003c/sup\u003e in prefrontal astrocytes or Ca\u003csup\u003e2+\u003c/sup\u003e responses in PV interneurons. To this end, hM3DGq or KATII\u003csup\u003eKD\u003c/sup\u003e-hM3DGq expressing AAV was co-injected with an AAV expressing the Ca\u003csup\u003e2+\u003c/sup\u003e sensor, GCaMP6s (astrocytes) or loxP(rev)jGCaMP8m (PV interneurons), into the PFC of adult C57BL6/N or PV\u003csup\u003eCre\u003c/sup\u003e (JAX:008069) mice respectively. To access the PFC, a right-angled microprism attached to a cranial window was implanted into the subdural space within the fissure opposite the site of injection\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMethodological details regarding the surgical procedure and training of mice, as well as the \u003cem\u003ein vivo\u003c/em\u003e two-photon imaging experiments including image acquisition, quantification and analyses are available in the \u003cb\u003eSupplementary Information\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTissue collection for postmortem analyses\u003c/h2\u003e \u003cp\u003eThe animals were deeply anesthetized with an overdose of pentobarbital (Esconarkon ad us. vet., Streuli Pharma AG, Switzerland) and transcardially perfused with ice-cold artificial cerebrospinal fluid (pH 7.4)\u003csup\u003e41, 49, 50\u003c/sup\u003e. The brains were immediately removed from the skull and either frozen on dry ice and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until further processing (molecular analyses of KATII expression) or postfixed in 4% PFA for 6 hrs before cryoprotection in 30% sucrose in PBS for 24\u0026ndash;48 hours, freezing on dry ice and storage at \u0026minus;\u0026thinsp;80\u0026deg;C until further processing.\u003c/p\u003e \u003cp\u003ePlasma from MIA and CON offspring was collected immediately prior to transcardial perfusion. The atrium was incised, and blood was collected into EDTA-coated blood collection tubes to prevent coagulation. Samples were centrifuged at 2000 \u0026times; g for 10 minutes to separate plasma, which was then aliquoted and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry and laser-scanning confocal microscopy\u003c/h2\u003e \u003cp\u003eImmunofluorescent staining and laser-scanning confocal microscopy were used to quantify the intensity of astrocyte markers in the PFC of CON and MIA offspring, the cellular expression pattern of the hM3DGq construct, and c-Fos expression in neuronal subtypes and astrocytes after chemogenetic activation of the latter.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eSample processing\u003c/strong\u003e \u003cp\u003eFixed brains were cut coronally with a sliding microtome at 30 \u0026micro;m (eight serial sections) and stored at \u0026minus;\u0026thinsp;20\u0026deg;C in cryoprotectant solution [50 mM sodium phosphate buffer (pH 7.4) containing 15% glucose and 30% ethylene glycol; Sigma-Aldrich, Switzerland] until further processing.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eImmunofluorescent staining\u003c/strong\u003e \u003cp\u003eImmunofluorescent stainings were performed according to established protocols\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Briefly, the brain sections were incubated with primary antibodies diluted in tris buffer containing 0.2% Triton X-100 and 2% normal serum free-floating under constant agitation (100 rpm) overnight at 4\u0026deg;C. The following day, sections were washed and incubated with secondary antibodies diluted in tris buffer containing 2% normal serum under constant agitation (100 rpm) for 30 min at room temperature. After incubation, which was shielded from light, the sections were washed, mounted onto gelatinized glass slides, coverslipped with Dako fluorescence mounting medium, and stored in the dark at 4\u0026deg;C until image acquisition.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eImage acquisition\u003c/strong\u003e \u003cp\u003eImmunofluorescence images were captured by laser scanning confocal microscopy or with Airyscan confocal microscopy (Zeiss LSM800 with Airyscan). To assess the selectivity of hM3DGq construct expression, 6 images randomly selected from 3 consecutive sections within the area of construct expression were acquired per animal using a 40\u0026times; (oil, NA 1.4) objective with a zoom of 0.45. Higher resolution image stacks for representative images of cell type specific expression of hM3DGq-mCherry were acquired in Airyscan mode using a 40\u0026times; lens, NA 1.4, oil and processed using the default settings provided by ZEN 2.6 blue edition software (Zeiss, Switzerland). For the c-Fos expression analyses in different cell types of the PFC, 6 randomly selected images within the area of construct expression across 3 consecutive sections were acquired per animal using a 25\u0026times; (oil, NA 0.8) objective with a zoom of 1. For the intensity analyses of astrocyte markers, 9 images randomly selected from 3 consecutive sections within the PFC were acquired per animal using a 25\u0026times; (oil, NA 0.8) objective with a zoom of 0.7. Imaging for each experiment were acquired on the same experimental day by an experimenter blinded to the experimental conditions, whereby imaging settings were kept constant throughout an entire imaging day.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eImage analyses\u003c/span\u003e: Image analyses were performed using the ImageJ software by an experimenter blinded to the experimental conditions. Intensity (mean grey value) per astrocyte marker was measured and calculated on z-projected images with a threshold applied to remove background. The mean intensity was than calculated over the 9 images for each animal. For the assessment of selective expression of the hM3DGq construct in astrocytes, the number of mCherry\u003csup\u003e+\u003c/sup\u003e, S100β\u003csup\u003e+\u003c/sup\u003e, NeuN\u003csup\u003e+\u003c/sup\u003e, mCherry\u003csup\u003e+\u003c/sup\u003e/ S100β\u003csup\u003e+\u003c/sup\u003e, and mCherry\u003csup\u003e+\u003c/sup\u003e/NeuN\u003csup\u003e+\u003c/sup\u003e cells was counted within each image. Expression selectivity was then calculated by dividing the number of colocalized cells with the number of total mCherry\u003csup\u003e+\u003c/sup\u003e cells and multiplied by 100. For the cell-type specific c-Fos mapping within the PFC, the number of c-Fos\u003csup\u003e+\u003c/sup\u003e cells, the number of target cells, and the number of co-localized cells were counted within each image. The percentage of c-Fos\u003csup\u003e+\u003c/sup\u003e cells was calculated as follows: (number of co-localized cells/number of target cells) * 100 for each image (referred to as field of view (FOV)). The mean % of c-Fos\u003csup\u003e+\u003c/sup\u003e cells, the mean number of target cells, and the mean number of c-Fos\u003csup\u003e+\u003c/sup\u003e cells were then calculated over the 6 images for each animal.\u003c/p\u003e \u003cp\u003eMethodological details regarding immunohistochemistry, image acquisition, and analyses are available in the \u003cb\u003eSupplementary Information\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of brain metabolites of the kynurenine pathway\u003c/h2\u003e \u003cp\u003eSamples of PFC bulk tissue (including anterior cingulate, prelimbic, and infralimbic cortices) were collected and processed as described in the \u003cb\u003eSupplementary Information\u003c/b\u003e. Brain metabolites of the KYN pathway were quantified using liquid chromatography-nanoelectrospray ionization tandem mass spectrometry as described in the \u003cb\u003eSupplementary Information\u003c/b\u003e. Besides KYN and KYNA, the following metabolites were quantified as well: tryptophan (TRP), quinolinic acid (QUIN), and 3 hydroxykynurenine (3-HK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of plasma kynurenine and kynurenic acid\u003c/h2\u003e \u003cp\u003ePlasma samples for KYN and KYNA measurements were diluted (1:2 v/v and 1:10 v/v, respectively) with ultrapure water to a final volume of 100 \u0026micro;l and acidified with 25 \u0026micro;l of 6% perchloric acid. After centrifugation (12,000 \u0026times; g, 10 min), metabolite levels were measured in 20 \u0026micro;l of the resulting supernatant by high-performance liquid chromatography with fluorimetric detection as described in the \u003cb\u003eSupplementary Information\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time PCR analysis\u003c/h2\u003e \u003cp\u003eQuantitative RT-PCR was used to measure \u003cem\u003emAadat\u003c/em\u003e RNA levels in PFC extracted from adult C57BL6/N mice expressing no construct (referred to as wild-type) and adult mice that express the KATIIKD-hM3DGq-HA construct in astrocytes. A mouse TaqMan gene expression assay for Aadat (assay ID: Mm00496169_m1, catalogue number: 4331182; Thermo Fisher Scientific, Zurich, Switzerland) was used. The samples were run in 384-well formats in triplicates as multiplexed reactions with the normalizing internal control (36B4). Relative gene expression was calculated with the 2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔCt method. All RT-PCRs and analyses were conducted by an experimenter blind to the experimental conditions. Relative changes in \u003cem\u003emAadat\u003c/em\u003e expression were finally compared to the average relative mAadat expression in the PFC of wild-type C57BL6/N mice. Methodological details regarding RNA extraction and TagMan qRT-PCR are available in the \u003cb\u003eSupplementary Information\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eWestern blot was used to investigate KATII protein levels in total PFC homogenates extracted from adult C57BL6/N male mice expressing no construct (referred to as wild-type) and adult male mice that express KATIIKD-hM3DGq-HA construct in astrocytes. Lysis and sample preparation were performed according to established protocols (see \u003cb\u003eSupplementary Information\u003c/b\u003e). Levels of KAT II (AADAT, rabbit, polyclonal, PA5-88974, Invitrogen,Switzerland, 1:700) were analyzed with reference to Histone 3 (H3, rabbit, monoclonal (D1H2), 4499, Cell Signaling, USA, 1:1000) as the housekeeping control. Methodological details regarding protein separation, transfer and detection are available in the \u003cb\u003eSupplementary Information\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analyses\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using SPSS Statistics (version 29.0, IBM, Armonk, NY, USA) and Prism (version 9.0; GraphPad Software, La Jolla, CA, USA). Statistical significance was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Detailed information regarding the statistical analyses used for each experiment is available in the \u003cb\u003eSupplementary Information\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCONTRIBUTIONS\u003c/h2\u003e \u003cp\u003eV.B., J.F., S.M.S., R.S., E.T., K.D.F., F.H., C.H., A.v.F.C., JC, U.W., and M.W. were involved in the acquisition, analysis, and interpretation of the study data; T.N. and U.M. were involved in the conception and design of the study and analysis and interpretation of the study data; T.N., A.S., S.B., B.W., and U.M. supervised research; T.N., U.M., and A.S. wrote the initial manuscript draft; all authors contributed to the reviewing and editing of the manuscript, and have given final approval for the version to be published.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eETHICS DECLARATION\u003c/strong\u003e \u003cp\u003eAll authors declare no competing interests. The funders of the study had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eACKNOWLEDGMENTS\u003c/h2\u003e \u003cp\u003eWe thank Jean-Charles Paterna and Lazaros Vasilikos from Viral Vector Facility (VVF) of the Neuroscience Center Zurich (ZNZ), Switzerland. We also thank Endre Laczko and Kurt Stefan Schauer from the Functional Genomics Center Zurich (FGCZ), Switzerland, for their technical assistance in liquid chromatography-nanoelectrospray ionization tandem mass spectrometry. This work was financially supported by the Swiss National Science Foundation (grant No. PZ00P3_202149 and grant No. P2ZHP3_174868 awarded to T.N.; grant No. 310030_188524, awarded to U.M.). Additional financial support was provided by the Brain \u0026amp; Behavior Research Foundation (grant No. 30963 awarded to T.N.), the Neuroscience Center Zurich (ZNZ PhD Grant 2022 awarded to T.N), the Olga-Mayenfisch Foundation (awarded to T.N.), and the UZH Candoc Grant, (grant No. FK-25-021 awarded to V.B.).\u003c/p\u003e\u003ch2\u003eDATA AVAILABILITY\u003c/h2\u003e \u003cp\u003eAll data are available in the main text or the supplementary materials. Any additional data that we inadvertently missed will be shared upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOliveira JF, Araque A (2022) Astrocyte regulation of neural circuit activity and network states. Glia 70:1455\u0026ndash;1466\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNagai J et al (2021) Behaviorally consequential astrocytic regulation of neural circuits. 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Mol Psychiatry 23:323\u0026ndash;334\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Astrocytes, cognition, kynurenine pathway, maternal immune activation (MIA), prefrontal cortex, parvalbumin interneuron","lastPublishedDoi":"10.21203/rs.3.rs-4492882/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4492882/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAstrocytes play critical roles in neural circuit function, but how they contribute to cognitive impairment remains poorly understood. Here, we identify astrocyte-derived kynurenic acid (KYNA), a neuroactive metabolite acting as endogenous N-methyl-D-aspartate receptor (NMDA) receptor antagonist, as a key mediator of cognitive dysfunction in the context of aberrant astrocyte activity. Using chemogenetic stimulation, pharmacological rescue, and astrocyte-specific knockdown of kynurenine aminotransferase II (KAT II), we show that elevated KYNA suppresses parvalbumin-positive interneuron activity in the prefrontal cortex, leading to disinhibition of pyramidal neurons and impairments in cognitive functions linked to cortical activity, including episodic-like and working memory as well as sensorimotor gating. These findings define an astrocyte-KYNA-interneuron axis that controls cortical excitability and cognition, linking glial metabolism to circuit imbalance and cognitive dysfunction with broad relevance to psychiatric and neurological disorders.\u003c/p\u003e","manuscriptTitle":"Astroglial disinhibition of cortical circuits disrupts cognition via kynurenic acid","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-30 08:58:30","doi":"10.21203/rs.3.rs-4492882/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"feee986c-89fb-4f3b-98aa-5aa041d15c6c","owner":[],"postedDate":"December 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":60120164,"name":"Biological sciences/Neuroscience/Glial biology/Astrocyte"},{"id":60120165,"name":"Biological sciences/Neuroscience/Diseases of the nervous system/Schizophrenia"},{"id":60120166,"name":"Biological sciences/Neuroscience/Diseases of the nervous system/Developmental disorders"}],"tags":[],"updatedAt":"2026-04-01T11:57:00+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-30 08:58:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4492882","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4492882","identity":"rs-4492882","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}