bioRxiv Subject Collection: Neuroscience's Journal

The 5-HT1A receptor antagonist WAY-100635 maleate reprograms metabolism to promote RGC differentiation and regeneration in retino-visual centers
Metabolic collapse of retinal ganglion cells (RGCs) onsets glaucoma, yet no approved drug directly protects these neurons. Through a live-cell mitochondrial screen in human stem-cell-derived hRGCs we uncovered WAY-100635 (WAY), a clinically tested 5-HT1A antagonist, as a systemic neuroprotectant. WAY triggers a reversible cyclic-AMP surge that activates PGC-1-driven reversible mitochondrial biogenesis and suppresses apoptosis. In glaucoma associated OPTNE50K hRGCs, WAY restores mitochondrial fitness, dampens excitotoxicity, and reprograms metabolism toward aerobic glycolysis, while in progenitors WAY boosts mitochondrial cristae maturation, oxidative phosphorylation, and cell-cycle exit to accelerate RGC specification. Daily intraperitoneal dosing preserves RGC bodies, neural activity, promotes axon regeneration into the optic nerve and vision centers after optic-nerve crush, as well as shows RGC protection and maintenance of visual acuity in chronic ocular hypertension glaucoma. As the non-invasive neuroprotective therapy with a human safety profile, WAY addresses a critical gap in glaucoma care and potentially for other mitochondrial optic neuropathies.

GRAPHICAL ABSTRACT

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Dynamic encoding of valence by the anterior cingulate cortex in mice
Fear is a double-edged sword: it supports survival based on learned associations between environmental cues and potential threats, but its dysregulation can lead to anxiety disorders and PTSD. Many studies have addressed the roles of the hippocampus and basolateral amygdala in the storage of the fear engram, but the role of the anterior cingulate cortex (ACC), especially during and immediately after fear acquisition, remains poorly defined. To address this gap, we longitudinally recorded ACC neuronal activity using single-photon calcium imaging in freely behaving adult male mice subjected to fear conditioning. Subjects acquired a conditioned freezing response to a tone cue (conditioned stimulus, CS) paired with light foot shocks (unconditioned stimulus, US), and ACC activity was monitored during cue pre-exposure, fear acquisition, fear recall, and fear extinction. Consistent with known functions of the ACC, neuronal responses were modulated by the US and by the novelty of the CS and US. Critically, both the number of CS-responsive neurons and the CS-associated population activity rose during acquisition, peaked during recall, and decreased throughout extinction. Neuronal populations responsive to the CS overlapped at a rate consistent with chance, suggesting that the ACC operates as a flexible integrative hub rather than containing stable engrams. Together, these findings indicate that ACC neuronal populations, but not engrams, represent novelty, pain, and the dynamic valence of a CS. Our findings are consistent with a model in which the ACC plays a role in threat appraisal and provides a learning signal that dynamically updates fear representations in other regions.

Predicting Brain Volumes from Anthropometric and Demographic Features: Insights from UK Biobank Neuroimaging Data
Brain size measures are well-studied and often treated as a confound in volumetric neuroimaging analyses. Yet their relationship with body anthropometric measures and demographics remains underexplored. In this study, we examined those relationships alongside age- and sex related differences in global brain volumes. Using brain magnetic resonance imaging (MRI) of healthy participants in the UK Biobank, we derived global measures of brain morphometry, including total intracranial volume (TIV), total brain volume (TBV), gray matter volume (GMV), white matter volume (WMV), and cerebrospinal fluid (CSF). We extracted these measures using the Computational Anatomy Toolbox (CAT) and FreeSurfer. Our analyses were structured in three approaches: across-sex analysis, sexspecific analysis, and impact of age analysis. Employing machine learning (ML), we found that TIV was strongly predicted by sex (across-sex = 0.68), reflecting sexual dimorphism. On the other hand, TBV, GMV, WMV, and CSF were more sensitive to age, with higher prediction accuracy when age was included as a feature, highlighting age-related changes in the brain structure, such as fluid expansion. Sex-specific models showed reduced TIV prediction ( {approx} 0.25) but improved TBV accuracy ( {approx} 0.44), underscoring sex-specific body-brain relationships. Anthropometrics enhanced prediction but only subsidiary to age and sex. These findings advance our understanding of brain-body scaling relationships and underscore the necessity of accounting for age and sex in neuroimaging studies of brain morphology.

Dendritic Polyglycerol Amine Substrate Extends the Viability of Mixed Glial Cultures for Repeated Isolation of Immature Oligodendrocyte Lineage Cells
Primary mixed glial cultures are key tools to isolate and study astrocytes, microglia and oligodendrocytes. Cell-substrate adhesion is critical for neural cell survival and differentiation. Cationic polymers like poly-D-lysine (PDL) are widely used to promote cell adhesion to cell culture substrates, however, PDL is not stable long-term, with cultured cells often detaching (peeling) after 2-3 weeks. Dendritic polyglycerol amine (dPGA) is a synthetic polycationic non-protein polymer biomimetic of poly-lysine that is highly resistant to degradation by cellular proteases. Substrates coated with dPGA promote cell adhesion and improve survival in long-term neuronal cultures. Here we assessed dPGA as a substrate coating to provide long-term support for mixed glial cultures. Oligodendrocyte precursor cells (OPCs) were isolated weekly by differential adhesion from cultures grown in T75 flasks with PDL or dPGA-coated substrates. Following two shake-off isolations, the cell layer in most PDL-coated flasks fully detached, rendering these flasks unusable for further culture. In contrast, dPGA-coated flasks consistently yielded cells for six or more sequential isolations over seven weeks in culture. dPGA-coated flasks produced more cells, a greater percentage of O4+ cells, and maintained similar proportions of OPCs and MBP-positive cells as when isolated from a PDL-coated substrate. dPGA is cyto-compatible, functionally superior, easy to use, low cost and a stable alternative to conventional cell substrate coatings. The enhanced long-term stability of mixed glial cultures grown on a dPGA substrate has the capacity to increase cellular yield, reduce animal use, and facilitate studies of oligodendrocyte cell biology.

Heterosynaptic interactions between dorsal and ventral hippocampus in individual medium spiny neurons of the nucleus accumbens ventromedial shell
Establishing learned associations between rewarding stimuli and the context under which those rewards are encountered is critical for survival. Hippocampal input to the nucleus accumbens (NAc) is a key connection involved in integrating environmental information and reward processing to facilitate goal-directed behaviors. This connection consists of two independent pathways originating from the dorsal (dHipp) or ventral (vHipp) hippocampus, which have previously been considered functionally and anatomically distinct. Here, we show overlap in dHipp and vHipp terminal fields in the NAc, which led us to reconsider this view and raise new questions regarding the potential interactions between dHipp and vHipp pathways in the NAc. Using optogenetics, electrophysiology, and transsynaptic labeling in adult male and female mice, we investigated anatomical and functional convergence of dHipp and vHipp in the NAc. We identified a subpopulation of dually innervated cells in the NAc medial shell where dHipp and vHipp inputs are located near one another along dendritic branches. We independently manipulated dHipp and vHipp inputs via two-color optogenetic manipulation during whole-cell electrophysiology recordings to confirm functional dual innervation of individual neurons and revealed heterosynaptic interactions between the two pathways. Altogether, these results demonstrate that dHipp and vHipp dually innervate a subset of neurons in the NAc, suggesting integration of these inputs at the level of individual neurons. Exploring the physiological and behavioral implications of this convergence will offer new insights into how individual neurons incorporate information from distinct inputs and how this integration may shape learning.

SIGNIFICANCE STATEMENTForming associations between rewards and the circumstances under which they are experienced is vital for survival. Hipp input to the NAc is essential for associating rewards with their environmental context to effectively guide motivated behaviors. This connection consists of two separate pathways originating from dHipp and vHipp that have long been considered distinct. Here, we reveal a subpopulation of neurons in the NAc shell innervated by both Hipp subregions as well as heterosynaptic interactions that occur between dHipp and vHipp synapses. These findings suggest that integration of distinct hippocampal information occurs at the single-neuron level, providing a critical mechanism underlying learning and motivated behavior while also opening new avenues for understanding how diverse contextual and reward signals shape decision-making.

Fundamental Sex Differences in Cocaine-Induced Plasticity of D1R- and D2R-MSNs in the Mouse Nucleus Accumbens Core
BACKGROUND: Previous studies have shown that cocaine-induced changes in nucleus accumbens shell (NAcSh) medium spiny neurons (MSNs) differ based on dopamine receptor subtype expression, the sex of the animal, and for females, phase of the estrous cycle. These findings highlight the need to account for both sex and estrous cycle when studying drug-mediated alterations in neurophysiology. Whether MSNs of the nucleus accumbens core (NAcC), which serve different aspects of addiction, will exhibit similar sex and estrous cycle effects with cocaine administration was investigated. METHODS: Mice underwent a 5-day locomotor sensitization paradigm via daily cocaine administration (15 mg/kg, s.c.) followed by a 1- to 4-day drug-free abstinence period. We examined NAcC MSN excitability by obtaining ex vivo whole-cell recordings from differentially labeled dopamine D1-receptor expressing MSNs (D1R-MSNs) and dopamine D2-receptor expressing MSNs (D2R-MSNs) obtained from male mice or female mice that were either in estrus or diestrus. RESULTS: In this genetic background of mice, both male and female mice sensitized to cocaine in a similar manner. In males, there were no cocaine-induced changes in D1R-MSN or D2R-MSN excitability, with D2R-MSNs exhibiting greater excitability. In saline-treated females, D1R-MSN excitability fluctuated across the estrous cycle with increased excitability during estrus. Following cocaine, estrous cycle-dependent D1R-MSN excitability was arrested, fixed at an intermediate value between estrus and diestrus when compared to saline controls. D2R-MSNs did not change either across the estrous cycle or following cocaine. When comparing MSN subtypes, in diestrus, D2R-MSNs were more excitable under saline conditions, but indistinguishable from D1R-MSNs following cocaine. In contrast, during estrus, D1R- and D2R-MSN excitability was similar in saline treated animals, but with cocaine, D2R-MSNs displayed heightened excitability. CONCLUSIONS: There are fundamental sex differences in cocaine-induced changes to the excitability of D1R-MSNs in the NAcC. After cocaine exposure, female mice in diestrus exhibited a significant main effect change in MSN excitability, an inversion of what had previously been demonstrated in the NAcSh where no cocaine-induced changes were observed. These data suggest that there are distinct differences in the neuropharmacological effect of cocaine in males versus females that are shell and core specific.

Abnormal Elevated Connectivity During Language Processing is Associated with Poor Cognitive Performance in Children with Self-limited Epilepsy with Centrotemporal Spikes
Self-Limited Epilepsy with Centrotemporal Spikes (SeLECTS) is associated with language impairments despite seizures originating in the motor cortex, suggesting aberrant cross-network interactions. Here we tested whether functional connectivity in SeLECTS during language tasks predicts language performance. We recorded high-density EEG from right-handed children with SeLECTS (n=31) and age-matched controls (n=32) during verb generation, repetition, and resting tasks. Phonological awareness was assessed using the Comprehensive Test of Phonological Processing-2. Connectivity between bilateral motor cortices and language regions (the left inferior frontal and superior temporal cortices and their right hemisphere homologues) was measured using weighted Phase Lag Index (wPLI). Children with SeLECTS demonstrated significantly elevated connectivity between motor and language regions during language processing. Motor-to-frontal connectivity was higher in SeLECTS during both verb generation and repetition tasks. Frontal-to-temporal connectivity was elevated specifically during verb generation. Higher interhemispheric connectivity (between the left and right hemispheres) during language tasks strongly predicted worse phonological awareness in children with SeLECTS ({beta} = -40 to -61, all p<0.005), but not controls. Together, we found that children with SeLECTS exhibited pathologically elevated connectivity between motor and language networks that was strongly associated with impaired phonological awareness. These findings identify aberrant interhemispheric connectivity as a pathophysiological mechanism underlying language dysfunction and establish EEG-based connectivity measures as a potential biomarker for guiding targeted neuromodulation therapies to treat cognitive impairments in pediatric epilepsy.

Lie Detectors for Face Recognition
Lie detection is important for government law enforcement. Current lie detection methods such as the polygraph test have been found to be unreliable (Meijer et al. 2017). New lie detection technology is currently arising that is based on fMRI; however, single subject tests have only been successful in detecting lies 88% of the time (Langleben et al., 2005; Wild, 2005). One of the main problems with most fMRI-based approaches is that they assume that various acts of deception involve common brain regions, (Ganis et al., 2003). In this work I propose a much more accurate fMRI lie detection method that does not make this assumption and is domain based. In my investigation, rather than trying to localize brain regions that are indicative of lying in general, I localize brain regions that indicate lying specifically about face recognition. In criminal investigations one frequently needs to establish familiar relations between a suspect, victim and/or witness. This type of information can be used as circumstantial evidence in a crime. In this work I propose to use fMRI to detect whether a suspect has any familiarity with an individual face. I find that activation in the left inferior frontal gyrus was a reliable discriminator for face familiarity.

High-resolution whole-brain magnetic resonance spectroscopic imaging in youth at risk for psychosis
Recent developments in acquisition and reconstruction of 3-dimensional magnetic-resonance spectroscopic imaging (3D-MRSI) enable the high-resolution mapping of multiple neurometabolites in the whole-brain, in vivo. Leveraging this capability, we created a voxel-based pipeline that corrects and spatially normalizes whole-brain maps of total N-acetylaspartate (tNAA), myo-inositol (Ins), choline compounds, glutamate + glutamine, and creatine + phosphocreatine. We examined a clinical cohort of adolescents and young adults at risk for psychosis (n= 21) --meeting DSM-5 criteria for Attenuated Psychosis Syndrome (APS)or Schizotypal Personality Disorder (SCZT)-- and age-/sex-matched healthy controls (n =13), as well as an independent non-clinical sample of adolescents (n = 61). We first aimed at assessing the reproducibility of MRSI measures across datasets and scanning sites, then validate the feasibility of a whole-brain voxel-based analyses on 3D-MRSI data and eventually test the sensitivity of this approach. Metabolite distributions showed reproducible regional variation in standard space between the two independent samples and scanning sites (r ranging from 0.82 to 0.99). Relative to controls, at-risk participants exhibited higher tNAA in frontal grey matter; the SCZT subgroup additionally displayed widespread cortical and subcortical Ins elevations compared with both APS and controls. Voxel-based analyses of structural (i.e., gray and white matter volumes or densities) and dijusion (i.e., generalized fractional anisotropy) parameters yielded no significant dijerences between risk participants and controls. These findings suggest the sensitivity of high-resolution 3D-MRSI for detecting subtle neurometabolic alterations at the group level in the early stages of psychotic disorders. Detailed brain metabolic mapping has the potential to help with early identification of young people at risk for psychosis or other mental disorders.

Multisensory integration for active mechanosensation in Drosophila flight
To support robust behaviors in highly variable environments, animals rely on active sampling of their sensory surroundings. Here, we use tethered, flying Drosophila melanogaster and a multisensory behavioral apparatus simulating forward flight to determine how visual and mechanosensory information are integrated and control active movements of an important multimodal sensory organ, the antennae. We found that flies perform active antennal movements in response to varying airflow, and that the direction of these movements changes depending on the visual environment. Next, we found that antennal movements are amplified in the presence of visual motion, but only when the fly was flying. Through mechanical and optogenetic manipulation of mechanosensory input, we found that mechanosensory feedback is vital to antennal positioning at flight onset. Additionally, we observed unexpected changes in wingbeat frequency when the antenna was mechanically stabilized, suggesting that multiple antennal mechanosensors contribute to flight regulation. Finally, we show that integration of mechanosensory and visual cues for controlling antennal motion follows in a "winner-takes-all" paradigm dependent on the stimulus frequency, mirroring visuo-mechanosensory guided behaviors in other species. Together, these results reveal novel behavioral gating of sensory information and expand our understanding of the efferent control of active sensing.

MAPseq2: a sensitive and cost-effective barcoded connectomics method
The barcoded connectomics tool MAPseq enables highly multiplexed projection mapping of individual neurons by translating neuroanatomy into a DNA sequencing problem. Here we present MAPseq2, a user-friendly protocol with 3-4 fold increased barcode detection sensitivity and ~10 fold decreased cost relative to the current MAPseq protocol. As MAPseq workflows are used across a range of barcoded connectomics methods, including BARseq, BRICseq, and ConnectID, all improvements in MAPseq2 directly transfer to these technologies.

Specific anterior-posterior brain-wide input patterns support specialized visuospatial processing in the mouse retrosplenial cortex
The retrosplenial cortex (RSC) is a key integrative hub involved in spatial orientation, navigation, and diverse cognitive and mnemonic processes. In rodents, RSC neurons carry rich sensory and navigational signals and are interconnected with sensory, motor, thalamic, and hippocampal circuits, supporting multimodal integration. However, the cellular circuit mechanisms that underlie this integration remain poorly understood. Here, we combined two-photon calcium imaging in navigating mice with brain-wide retrograde tracing to investigate how visual and positional signals are encoded and distributed across the RSC, with functional data covering both the medial-to-lateral and anterior-to-posterior dimensions of the RSC. We found that anterior and posterior RSC neurons exhibit distinct encoding properties and afferent connectivity. While anterior RSC neurons preferentially encode high-speed visual motion and display sharp, reliable position tuning, posterior RSC neurons are more responsive to low-speed stimuli and exhibit broader, weaker position-related activity. These functional specializations are paralleled by distinct long-range input patterns: anterior RSC receives dense projections from motor, parietal, and hippocampal-related areas, regions associated with strong position-related signals, whereas posterior RSC is more strongly innervated by visual cortices. Together, our results reveal a topographic organization of visual and navigational signals in the RSC, with anatomically distinct subregions potentially supporting different roles in visuospatial integration during navigation.

A connectomics-based analysis of lamina monopolar cells reveals mechanisms of spatial processing in insect motion vision
Many animals strongly rely on their sense of vision, as it provides information about the natural world with particularly high dimensionality. In insects, the first visual processing stage of the brain, the lamina, plays an important role in parallel processing of this complex information. Its main relay neurons, lamina monopolar cells (LMCs), receive information directly from the photoreceptors and shape the contrast, luminance, spatial and temporal tuning of the insect visual system in a cell-type specific manner. One of their best-investigated downstream targets is the motion vision pathway. However, how LMC types that feed into motion processing delineate contrast and luminance is only known from fruit flies, while the contribution of LMCs to spatial processing has only been described in hawkmoths. Here, we provide a novel characterization of hawkmoths lamina monopolar cells, to integrate the contrast, luminance and spatial processing properties of LMCs in the motion pathway. We used serial block-face scanning electron microscopy to reconstruct the anatomical fine structure of LMCs in a focal lamina cartridge, including their pre- and post- synaptic connections. Combining our novel LMC classification with intracellular recordings, we further investigated the functional role of hawkmoth L1 and L2 in terms of contrast and spatial processing. We show that unlike in flies, L1 and L2, the main relay neurons to the motion pathway, process contrast and luminance information in a similar manner. We further demonstrated that the spatial processing properties of these cells are highly similar as well, and can be explained by the density and the distribution of their synapses across different lamina layers. Based on these findings, we propose that the different lamina layers support distinct connectivity and functional roles in spatial processing.

Two-Point Calibration Protocol for the FRET Indicator Pyronic in Neurons
Significance: Pyruvate is a nodal intermediate in cellular metabolism, positioned at the crossroads between glycolysis and fermentative metabolism. It is exchanged between the intracellular and extracellular compartments through the proton-coupled monocarboxylate transporters and between the cytosol and mitochondria through the mitochondrial pyruvate carrier, where it serves as a primary carbon source for respiration. Aim: Our goal is to present a detailed protocol for quantifying cytosolic pyruvate concentration in neurons at single-cell resolution using a minimally invasive, two-point calibration approach with the FRET-based genetically-encoded fluorescent indicator Pyronic. Approach: This protocol is based on a non-invasive pharmacological two-point calibration approach, where Pyronic s dynamic range ({Delta}RMAX) is established by using trans-acceleration exchange to deplete intracellular pyruvate (RMIN), and by inducing Pyronic saturation (RMAX) through the combination of inhibition of pyruvate export, stimulation of its production, and blockade of its mitochondrial consumption. The protocol also incorporates the previously published KD values for Pyronic obtained from in vitro experiments. This procedure does not require the use of detergents to permeabilize the cells. Results: Implementing this protocol enables the measurement of absolute cytosolic pyruvate concentrations. This quantitative parameter facilitates comparisons of pyruvate metabolism across different cells, samples and experimental batches, thereby enabling the comparison between a plethora of experimental conditions. Conclusions: The FRET-based fluorescent indicator Pyronic can be reliably calibrated using a minimally invasive, pharmacology-based two-point calibration protocol in neurons, thus providing a robust and quantitative method to study pyruvate metabolism under various physiological and pathological scenarios.

The origins of time: a systematic review of the neural signatures of temporal prediction in infancy
From birth, individuals' interpersonal dimension is underpinned by progressive learning of social interaction rules, their variations rooted in the temporal prediction of sensory events, and the inferences made about the organization of the social world. How this dimension is structured during infancy and articulated at the neural level is a critical question for cognitive and affective neurosciences. This systematic review aims to define the neural signatures of temporal prediction in newborns and infants and to discuss them in the context of the development of proximal cognitive and affective neural functions. Eight peer-reviewed studies were included, with 228 infants from birth to 9 months of age. Studies have evidenced that neural signatures of temporal prediction in infants present a broad cerebral localization, including the anterior and medial parts of the brain, especially in the frontal and central areas. Temporal prediction mechanisms emerge well before birth and evolve from early sensory-driven responses to complex top-down processing within the first year, shaped by both innate and experience-dependent factors, with influences like wakefulness and musical exposure that modulate neural integration across sensory and higher-order brain regions.

Attention is all you need (in the brain): semantic contextualization in human hippocampus
In natural language, word meanings are contextualized, that is, modified by meanings of nearby words. Inspired by self-attention mechanisms in transformer-based large language models (LLMs), we hypothesized that contextualization in the brain results from a weighted summation of canonical neural population responses to words with those of the words that contextualize them. We examined single unit responses in the human hippocampus while participants listened to podcasts. We first find that neurons encode the position of words within a clause, that they do so at multiple scales, and that they make use of both ordinal and frequency-domain positional encoding (which are used in some transformer models). Critically, neural responses to specific words correspond to a weighted sum of that words non-contextual embedding and the embedding of the words that contextualize it. Moreover, the relative weighting of the contextualizing words is correlated with the magnitude of the LLM-derived estimates of self-attention weighting. Finally, we show that contextualization is aligned with next-word prediction, which includes prediction of multiple possible words simultaneously. Together these results support the idea that the principles of self-attention used in LLMs overlap with the mechanisms of language processing within the human hippocampus, possibly due to similar prediction-oriented computational goals.