bioRxiv Subject Collection: Neuroscience's Journal

Identification of neuron-glia signaling feedback in human schizophrenia using patient-derived, mix-and-match forebrain assembloids
Although abnormal activities across multiple cell types are believed to contribute to the development of various neurodevelopmental disorders, current brain organoid technologies fall short in accurately modeling the dynamic cellular interactions in the human brain. Recently, we developed a cellular reconstitution technology to create human forebrain assembloids with enhanced cellular diversity, representing dynamic interactions between neurons and glial cells. Here, we created patient-derived, mix-and-match forebrain assembloids, in which neurons, astrocytes, and microglia from both healthy individuals and schizophrenia patients were reconstituted in a combinatorial manner, and identified aberrant cellular interactions between neurons and glial cells in human schizophrenia. At the early stage, schizophrenia forebrain assembloids showed premature neurogenesis induced by the abnormal proliferation and differentiation of neural progenitor cells. Integrated modular analysis of gene expression in post-mortem schizophrenia brain tissue and brain assembloids found increased expression of tumor protein p53 (TP53) and nuclear factor of activated T-cells 4 (NFATC4), which functioned as master transcriptional regulators to epigenetically reprogram the transcriptome involved in the cellular dynamics of neuronal progenitor cells, leading to premature neurogenesis. At the later stage, we observed weakened structures of laminar organization of the cortical layers in forebrain assembloids and identified the neuron-dependent transcriptional plasticity of glial cells and their altered signaling feedback with neurons, in which neuronal urocortin (UCN) and protein tyrosine phosphatase receptor type F (PTPRF) elicited the expression of Wnt family member 11 (WNT11) and thrombospondin 4 (THBS4) in astrocytes and microglia, respectively. These aberrant signaling axes altered the neuronal transcriptome associated with neuronal response to various stimuli and synthetic processes of biomolecules, resulting in reduced synapse connectivity. Thus, we elucidated developmental stage-specific, multifactorial mechanisms by which dynamic cellular interplay among neural progenitor cells, neurons, and glial cells contribute to the development of the human schizophrenia brain. Our study further demonstrated the potential and applicability of patient-derived forebrain assembloid technology to advance our understanding of the pathogenesis of various neurodevelopmental disorders.

Sensory expectations shape neural population dynamics in motor circuits
The neural basis of movement preparation has been extensively studied during self-initiated actions where motor cortical activity during preparation shows a lawful relationship to the parameters of the subsequent action. However, movements are regularly triggered and constantly corrected based on sensory inputs caused by disturbances to the body or environment. Since such disturbances are often predictable and since preparing for disturbances would make movements better, we hypothesized that expectations about sensory inputs also influence preparatory activity in motor circuits. Here we show that when humans and monkeys are probabilistically cued about the direction of a future mechanical perturbation, they incorporate sensory expectations into their movement preparation and improve their corrective responses. Using high-density neural recordings, we establish that sensory expectations are widespread across the brain, including the motor cortical areas involved in preparing self-initiated actions. The geometry of these preparatory signals in the neural population state is simple, scaling directly with the probability of each perturbation direction. After perturbation onset, a condition-independent perturbation signal shifts the neural state leading to rapid responses that initially reflect sensory expectations. Based on neural networks coupled to a biomechanical model of the arm, we show that this neural geometry emerges through training, but only when the incoming sensory information indicating perturbation direction coincides with - or is preceded by - a condition-independent signal indicating that a perturbation has occurred. Thus, motor circuit dynamics are shaped by future sensory inputs, providing clear empirical support for the idea that movement is governed by the sophisticated manipulation of sensory feedback.

Red-shifted GRAB acetylcholine sensors for multiplex imaging in vivo
The neurotransmitter acetylcholine (ACh) is essential in both the central and peripheral nervous systems. Recent studies highlight the significance of interactions between ACh and various neuromodulators in regulating complex behaviors. The ability to simultaneously image ACh and other neuromodulators can provide valuable information regarding the mechanisms underlying these behaviors. Here, we developed a series of red fluorescent G protein-coupled receptor activation-based (GRAB) ACh sensors, with a wide detection range and expanded spectral profile. The high-affinity sensor, rACh1h, reliably detects ACh release in various brain regions, including the nucleus accumbens, amygdala, hippocampus, and cortex. Moreover, rACh1h can be co-expressed with green fluorescent sensors in order to record ACh release together with other neurochemicals in various behavioral contexts using fiber photometry and two-photon imaging, with high spatiotemporal resolution. These new ACh sensors can therefore provide valuable new insights regarding the functional role of the cholinergic system under both physiological and pathological conditions.

Association of Bile Acids with Connectivity of Executive Control and Default Mode Networks in Patients with Major Depression
Objective: Bile acids may contribute to pathophysiologic markers of Alzheimers disease, including disruptions of the executive control network (ECN) and the default mode network (DMN). Cognitive dysfunction is common in major depressive disorder (MDD), but whether bile acids impact these networks in MDD patients is unknown. Methods: Resting state functional magnetic resonance imaging (fMRI) scans and blood measures of four bile acids from 74 treatment-naive adults with MDD were analyzed. Dorsolateral prefrontal cortex (DLPFC) seeds were used to examine connectivity of the ECN and posterior cingulate cortex (PCC) seeds were used for the DMN. Using a whole-brain analysis, the functional connectivity of these seeds was correlated with serum levels chenodeoxycholic acid (CDCA) and its bacterially-derived secondary bile acid, lithocholic acid (LCA). Results: CDCA levels were strongly and inversely correlated with connectivity between DLPFC regions of the ECN (R2= .401, p<.001). LCA levels were strongly and positively correlated with connectivity of the DLPFC and left inferior temporal cortex of the ECN (R2=.263, p<.001). The LCA/CDCA ratio was strongly and positively correlated with connectivity of the DLPFC with two components of the ECN: bilateral inferior temporal cortex and the left superior and inferior parietal lobules (all R2>.24, all p<.001). For the DMN, the LCA/CDCA ratio was strongly and negatively correlated with connectivity of the PCC with multiple bilateral insula regions (all R2>0.25, all p<.001). Conclusions: The relationship between LCA and CDCA levels and functional connectivity of the ECN and DMN suggests potential shared pathophysiologic processes between Alzheimers disease and MDD.

In vivo multiplex imaging of dynamic neurochemical networks with designed far-red dopamine sensors
Neurochemical signals like dopamine (DA) play a crucial role in a variety of brain functions through intricate interactions with other neuromodulators and intracellular signaling pathways. However, studying these complex networks has been hindered by the challenge of detecting multiple neurochemicals in vivo simultaneously. To overcome this limitation, we developed a single-protein chemigenetic DA sensor, HaloDA1.0, which combines a cpHaloTag-chemical dye approach with the G protein-coupled receptor activation-based (GRAB) strategy, providing high sensitivity for DA, sub-second response kinetics, and an extensive spectral range from far-red to near-infrared. When used together with existing green and red fluorescent neuromodulator sensors, Ca2+ indicators, cAMP sensors, and optogenetic tools, HaloDA1.0 provides high versatility for multiplex imaging in cultured neurons, brain slices, and behaving animals, facilitating in-depth studies of dynamic neurochemical networks.

FGF21 Analogue PF-05231023 on Alcohol Consumption and Neuronal Activity in the Nucleus Accumbens
Fibroblast growth factor 21 (FGF21) is a liver-derived hormone known to suppress alcohol consumption in mice and non-human primates. However, the role of FGF21 in modulating environmental and behavioural factors driving alcohol consumption, such as cue-driven responses and effortful actions to obtain alcohol, and its effects on neural activity related to consumption, remain unclear. Here, we evaluated the impact of PF-05231023, a long-acting FGF21 analogue, across multiple dimensions of alcohol consumption and motivation. PF-05231023 reduced alcohol intake and preference in a dose- and sex-specific manner; diminished approach behaviours following an alcohol but not sucrose cue; and decreased lever-pressing under a progressive-ratio schedule, both alone and when combined with the GLP-1 agonist Exendin-4. Additionally, PF-05231023 altered the microstructure of alcohol consumption by shortening drinking bouts and increased the recruitment of nucleus accumbens (Acb) neurons associated with bout termination. These findings demonstrate that PF-05231023 broadly suppresses alcohol-motivated behaviours and that targeting FGF21 signaling in combination with GLP-1 agonists may enhance therapeutic efficacy. Mechanistically, the observed reductions in alcohol consumption following PF-05231023 appear to involve diminished alcohol palatability and modulation of neuronal activity from distinct subsets of Acb neurons.

Navigating the Maze: Identifying Potential Pitfalls in Attention State Classification from fMRI Brain Patterns
Multi-voxel pattern analysis (MVPA) is a powerful technique to decode brain states from functional magnetic resonance imaging (fMRI) activity patterns. In neurofeedback (NF) applications, it has been used to perform real-time classification of brain activity patterns, establishing a closed-loop system that provides immediate feedback to the participants, enabling them to learn to control a complex mental state. However, MVPA has many potential limitations when applied to fMRI datasets (especially in real-time analysis) arising from small effect sizes, small number of training samples, high dimensionality of the data and, more generally, design choices. All these factors might produce inaccurate classification results. In this work, we followed a previous NF paradigm for sustained attention training. Participants were presented with composite images superimposing faces and scenes. They were instructed to focus on one class (either face or scene) for an extended period. A logistic regression classifier was trained to determine whether participants were adequately focusing on the instructed category based on their fMRI data. We analysed the classification outputs of the no-feedback training runs using various classifier settings, including whole brain data and different masking approaches, combined with different methods for the computation of single-trial fMRI responses. Furthermore, a ventricle mask was used as a control condition for the classification task, and simulations were carried out to assess the influence of the class order on the classification performances. We found inflation of the decoding accuracy for several common design choices and confounders. In particular, motion artefacts and low frequency drifts coupled with the task timing might have artificially increased the accuracy scores. Furthermore, the simulations revealed that fixed order in the presentation of experimental conditions resulted in further inflation of the classification accuracies especially in GLM-based and average-based trial estimate methods. We discuss the drawbacks of applying MVPA using the analysed sustained attention paradigm and provide insights for future improvements.

Tumor-associated macrophages enhance tumor innervation and spinal cord repair
Tumor-associated macrophages (TAM) enhance cancer progression by promoting angiogenesis, extracellular matrix (ECM) remodeling, and immune suppression. Nerve infiltration is a hallmark of various cancers and is known to directly contribute to tumor growth. However, the role of TAM in promoting intratumoral nerve growth remains poorly understood. In this study, we demonstrate that TAM expressed a distinct ''neural growth'' gene signature. TAM actively enhance neural growth within tumors and directly promote neurites outgrowth. We identify secreted phosphoprotein 1 (Spp1) as a key mediator of TAM-driven neural growth activity, which triggers neuronal mTORC2 signaling. Leveraging this new neural growth function, which added to the TAM wound healing properties, we explored TAM potential to repair central nervous system. Adoptive transfer of in vitro-generated TAM in a severe complete-compressive-contusive spinal cord injury (scSCI) model, not only repaired the damaged neural parenchyma by improving tissue oxygenation, ECM remodeling, and dampening chronic inflammation, but also resulted in neural regrowth and partial functional motor recovery. Proteomic analysis and subsequent functional validation confirmed that TAM-induced spinal cord regeneration is mediated through the activation of neural mTORC2 signaling pathways. Collectively, our data unveil a previously unrecognized role of TAM in tumor innervation, neural growth, and neural tissue repair.

Impact of dopaminergic modulation on the state transitions of striatal medium spiny neuron sub-types - a computational study
Medium spiny neurons (MSN) of the striatum are known for their bistable membrane potential leading to two states - a hyperpolarized down-state and relatively depolarized up-state. Glutamatergic inputs from the hippocampus play a key role in switching the cell to up-state. This gating is known to play a key role in regulating when other synaptic inputs, such as from the cortex, should generate action potentials and when they should be considered as noise. Therefore, it is crucial for the transition between states to occur normally as abnormal gating has implications in conditions such as Schizophrenia. Although dopamine is reported to modulate ion channels of MSN sub-types - dMSN and iMSN, its influence on the state transition times and up-state dwell times are being reported here. This was done using biophysically constrained spiny models of dMSN and iMSN with explicit dopamine receptors. Additionally, strong correlation between state transition times and spiking frequencies of MSN sub-types was observed.

Sexual Failure Decreases Sweet Taste Perception in Male Drosophila via Dopaminergic Signaling
Sweet taste perception, a critical aspect of the initiation of feeding behavior, is primarily regulated by an animal's internal metabolic state. However, non-metabolic factors, such as motivational and emotional states, can also influence peripheral sensory processing and hence feeding behavior. While mating experience is known to induce motivational and emotional changes, its broader impact on other innate behaviors such as feeding remains largely uncharacterized. In this study, we demonstrated that mating failure of male fruit flies suppressed sweet taste perception via dopamine signaling in specific neural circuitry. Upon repetitive failure in courtship, male flies exhibited a sustained yet reversible decline of sweet taste perception, as measured by the proboscis extension reflex (PER) towards sweet tastants as well as the neuronal activity of sweet-sensing Gr5a+ neurons in the proboscis. Mechanistically, we identified a small group of dopaminergic neurons projecting to the subesophageal zone (SEZ) and innervating with Gr5a+ neurons as the key modulator. Repetitive sexual failure decreased the activity of these dopaminergic neurons and in turn suppressed Gr5a+ neurons via Dop1R1 and Dop2R receptors. Our findings revealed a critical role for dopaminergic signaling in integrating reproductive experience with appetitive sensory processing, providing new insights into the complex interactions between different innate behaviors and the role of brain's reward systems in regulating internal motivational and emotional states.

Engineered biological neural networks as basic logic operators
We present an in vitro neuronal network with controlled topology capable of performing basic Boolean computations, such as NAND and OR. Neurons cultured within polydimethylsiloxane (PDMS) microstructures on high-density microelectrode arrays (HD-MEAs) enable precise interaction through extracellular voltage stimulation and spiking activity recording. This system allows for the investigation of input-output relationships that define non-linear biological activation functions. Additionally, we analyze various output encoding schemes, comparing the limitations of rate coding with the potential advantages of spike-timing-based coding strategies. This work contributes to the advancement of hybrid intelligence and biocomputing by offering insights into neural information encoding and decoding with the potential to directly validate bio-inspired computational mechanisms used in artificial intelligence (AI) systems.

Cortical projection neurons with distinct axonal connectivity employ ribosomal complexes with distinct protein compositions
Diverse subtypes of cortical projection neurons (PN) form long-range axonal projections that are responsible for distinct sensory, motor, cognitive, and behavioral functions. Translational control has been identified at multiple stages of PN development, but how translational regulation contributes to formation of distinct, subtype-specific long-range circuits is poorly understood. Ribosomal complexes (RCs) exhibit variations of their component proteins, with an increasing set of examples that confer specialized translational control. Here, we directly compare the protein compositions of RCs in vivo from two closely related cortical neuron subtypes-cortical output "subcerebral PN" (SCPN) and interhemispheric "callosal PN" (CPN)- during establishment of their distinct axonal connectivity. Using retrograde labeling of subtype-specific somata, purification by fluorescence-activated cell sorting, ribosome immunoprecipitation, and ultra-low-input mass spectrometry, we identify distinct protein compositions of RCs from these two subtypes. Strikingly, we identify 16 associated proteins reliably and exclusively detected only in RCs of SCPN. 10 of these proteins have known interaction with components of ribosomes; we further validated ribosome interaction with protein kinase C epsilon (PRKCE), a candidate with roles in synaptogenesis. PRKCE and a subset of SCPN-specific candidate ribosome-associated proteins also exhibit enriched gene expression by SCPN. Together, these results indicate that ribosomal complexes exhibit subtype-specific protein composition in distinct subtypes of cortical projection neurons during development, and identify potential candidates for further investigation of function in translational regulation involved in subtype-specific circuit formation.

Aggregation promoting sequences rather than phosphorylation are essential for Tau-mediated toxicity in Drosophila
Background: Disease-modifying therapies for tauopathies like Alzheimer's disease have targeted Tau hyperphosphorylation and aggregation, as both pathological manifestations are implicated in Tau-mediated toxicity. However, the relative contributions of these pathology-linked changes to Tau neurotoxicity remain unclear. Methods: Leveraging the genetic tractability of Drosophila, we generated multiple inducible human Tau transgenes with altered phosphorylation status and/or aggregation propensity. Their individual and combined impact was tested in vivo by quantifying Tau accumulation and neurodegenerative phenotypes in the aging fly nervous system. Results: We report that phospho-mimicking Tau (hTau2N4RE14) induced profound neurodegeneration, supporting a neurotoxic role for phosphorylation. However, when we rendered hTau2N4RE14 aggregation incompetent, by deleting the 306VQIVYK311 motif in the microtubule-binding region, neurotoxicity was abolished. Moreover, a peptide inhibitor targeting this motif efficaciously reduced Tau toxicity in aging Drosophila. Conclusion: Neurodegeneration mediated by Tau hyperphosphorylation is gated via at least one aggregation-mediating motif on the protein. This highlights the primacy of blocking Tau aggregation in therapy, perhaps without the need to clear phosphorylated species.

Balancing Act: A Neural Trade-Off Between Coherence and Creativity in Spontaneous Speech
Effective communication involves a delicate balance between generating novel, engaging content and maintaining a coherent narrative. The neural mechanisms underlying this balance between coherence and creativity in discourse production remain unexplored. The aim of the current study was to investigate the relationship between coherence and creativity in spontaneous speech, with a specific focus on the interaction among three key neural networks: the Default Mode Network, Multiple Demand Network, and the Semantic Control Network. To this end, we conducted a two-part analysis. At the behavioural level, we analysed speech samples produced in response to topic cues, computing measures of global coherence (indexing the degree of connectedness to the main topic) and Divergent Semantic Integration (DSI; reflecting the diversity of ideas incorporated in the narrative). Coherence and divergence in speech were negatively correlated, suggesting a trade-off between maintaining a coherent narrative structure and incorporating creative elements. At the neural level, higher global coherence was associated with greater activation in the Multiple Demand Network, emphasising its role in organising and sustaining logical flow in discourse production. In contrast, functional connectivity analyses demonstrated that higher DSI was related to greater coupling between the Default Mode and Multiple Demand Networks, suggesting that creative speech relies on a dynamic interplay between associative and executive processes. These results provide new insights into the cognitive and neural processes underpinning spontaneous speech production, highlighting the complex interplay between different brain networks in managing competing demands of being coherent and creative.

Network Segregation During Episodic Memory Shows Age-Invariant Relations with Memory Performance From 7 to 82 Years
Lower episodic memory capability, as seen in development and aging compared with younger adulthood, may partly depend on lower brain network segregation. Here, our objective was twofold: (1) test this hypothesis using within- and between-network functional connectivity (FC) during episodic memory encoding and retrieval, in two independent samples (n=734, age 7-82 years). (2) Assess associations with age and the ability to predict memory comparing task-general FC and memory-modulated FC. In a multiverse-inspired approach, we performed tests across multiple analytic choices. Results showed that relationships differed based on these analytic choices, were often weak, and mainly present in the cohort with the most data. Significant relationships indicated that (i) memory-modulated FC predicted memory performance and associated with memory in an age-invariant manner. (ii) In line with the so-called neural dedifferentiation view, task-general FC showed lower segregation with higher age in adults which was associated with worse memory performance. In development, although there were only weak signs of a neural differentiation, that is, gradually higher segregation with higher age, we observed similar lower segregation-worse memory relationships. This age-invariant relationships between FC and episodic memory suggest that network segregation is pivotal for memory across the healthy lifespan.

Investigating the Role of Glyoxalase 1 as a Therapeutic Target for Cocaine and Oxycodone Use Disorder
Methylglyoxal (MG) is an endogenously produced non-enzymatic side product of glycolysis that acts as a partial agonist at GABAA receptors. MG that is metabolized by the enzyme glyoxalase-1 (GLO1). Inhibition of GLO1 increases methylglyoxal levels, and has been shown to modulate various behaviors, including decreasing seeking of cocaine-paired cues and ethanol consumption. The goal of these studies was to determine if GLO1 inhibition could alter cocaine- or oxycodone-induced locomotor activation and/or conditioned place preference (CPP) to cocaine or oxycodone. We used both pharmacological and genetic manipulations of GLO1 to address this question. Administration of the GLO1 inhibitor s-bromobenzylglutathione cyclopentyl diester (pBBG) did not alter the locomotor response to cocaine or oxycodone. Additionally, pBBG had no significant effect on place preference for cocaine or oxycodone. Genetic knockdown of Glo1, which is conceptually similar to pharmacological inhibition, did not have any significant effects on cocaine place preference, nor did Glo1 overexpression affect locomotor response to cocaine. In summary, our results show that neither pharmacological nor genetic manipulations of GLO1 influence locomotor response or CPP to cocaine or oxycodone.

Grin2a Ablation Distinctly Reshapes PV+ and SST+ Interneuron circuits in the Medial Prefrontal Cortex to Amplify Gamma Oscillations
Schizophrenia (SCZ) is a debilitating mental health disorder marked by cognitive deficits, especially in executive functions, which are often resistant to treatment. The GRIN2A gene encodes the GluN2A subunit of N-methyl-D-aspartate (NMDA) receptor. Rare coding mutations in GRIN2A, such as protein-truncating variants, increase susceptibility to SCZ by ~20-fold. However, the effects on network dynamics and the function of GABAergic interneurons (INs) in the medial prefrontal cortex (mPFC) remain unclear. In this study, we use brain slices from Grin2a mutant mice to investigate inhibitory synaptic and oscillatory activity driven by parvalbumin-positive (PV+) and somatostatin-positive (SST+) INs. Our findings reveal significant alterations in synaptic currents and inhibitory modulation arise from GluN2A deficiency. Specifically, both heterozygous and knockout Grin2a mutants exhibit impaired synaptic transmission and release properties of INs inputs, including changes in release probability and evoked quantal events to layer V pyramidal neurons. Genotype-dependent alterations were observed in the frequency, kinetics, and amplitude of excitatory and inhibitory postsynaptic currents. Immunohistochemical analysis showed an increased density of PV+ and SST+ INs in the Grin2a mutants, consistent with an overall increase in inhibitory tone. Additionally, optogenetic stimulation revealed a dynamic shift for inducing gamma-band oscillations through PV+ INs. Overall, our study highlights the essential role of GluN2A-containing NMDA receptors in modulating inhibitory tone and maintaining network stability in the mPFC of adult mice and elucidates a pathophysiological mechanism that might contribute to cognitive deficits often observed in SCZ patients.

Critical Scaling of Novelty in the Cortex
The ability to detect unanticipated, novel events and rapidly relay this information across neural networks is fundamental to brain function, enabling the selection of appropriate behavioral responses. Here, we examine the transmission of holographically triggered action potentials in primary visual cortex of quietly resting mice, focusing on the dynamics of communication from pyramidal neurons. We demonstrate that these novel action potentials, which are uncorrelated with preceding activity, exert a disproportionally large influence on neighboring neurons. Their influence scales robustly to an exponent between 0.2 and 0.3 relative to their number. Remarkably, even a small number of novel action potentials can engage a majority of the local network, achieving high decoding accuracy of the perturbation origin in the face of high trial-by-trial variability and ongoing activity characterized by scale-invariant, parabolic neuronal avalanches. This heightened susceptibility to small, local perturbations aligns with the behavior of complex systems exhibiting critical dynamics. Our findings reveal that scaling underpins the efficient communication of unanticipated action potentials, suggesting it is a fundamental mechanism for detecting and processing novel events in the brain. These results provide new insights into the neural basis of novelty detection and highlight the importance of critical dynamics in cortical network function.