bioRxiv Subject Collection: Neuroscience's Journal

Mice dynamically adapt to opponents in competitive multi-player games
Competing for resources in dynamic social environments is fundamental for survival, and requires continuous monitoring of both 'self' and 'others' to guide effective choices. Yet our understanding of value-based decision-making comes primarily from studying individuals in isolation, leaving open fundamental questions about how animals adapt their strategies during social competition. Here, we developed an ethologically relevant multi-player game, in which freely-moving mice make value-based decisions in a competitive spatial foraging task. We found that mice integrate real-time spatial information about 'self' and the opponent to flexibly shift their preference towards safer, low-payout options when appropriate. Analyses of mice and reinforcement learning agents reveal that these behavioural adaptations cannot be explained by simple reward learning, but are instead consistent with optimal decision strategies guided by opponent features. Using a dynamical model of neural activity, we found that in addition to opponent effects, decisions under competition were also noisier and more sensitive to initial conditions, generating testable predictions for neural recordings and perturbations. Together, this work reveals a fundamental mechanism for competitive foraging, and proposes novel quantitative frameworks towards understanding value-based decision-making in a fast-changing social environment.

Top-down control of the descending pain modulatory system drives placebo analgesia
In placebo analgesia, prior experience and expectations lead to pain suppression by the administration of an inert substance, but causal evidence for its neural basis is lacking. To identify the underlying neural circuits, we reverse-translated a conditioned placebo protocol from humans to mice. Surprisingly, the placebo effect suppresses both nociception and unconditioned emotional-motivational pain-related behavior. Descending pain modulatory neurons in the periaqueductal gray (PAG) are critical for both morphine and placebo antinociception. The placebo effect depends on input to the PAG from the medial prefrontal and anterior cingulate cortices, but not anterior insular cortex. Conditioning enhances noxious stimulus-evoked endogenous opioid release in the PAG to produce analgesia. Our results suggest that cortical control of the descending pain modulatory system (DPMS) is gated by rapid endogenous opioid signaling in the PAG during placebo trials. This study bridges clinical and preclinical research, establishing a central role for the DPMS in placebo analgesia.

Transient Acute Neuronal Activation Response Caused by High Concentrations of Oligonucleotides in the Cerebral Spinal Fluid
Oligonucleotide (ON) therapeutics are promising as a disease-modifying therapy for central nervous system disorders. Intrathecal ON administration into the cerebral spinal fluid is a safe and effective delivery mode to the CNS. However, preclinical studies have shown acute toxicities following high-dose central ON delivery. Here we characterize a transient neurobehavioral change peaking 15 minutes after ON dosing and resolving after 120 minutes. Symptoms include shaking, muscle twitching, cramping, hyperactivity, stereotypic movements, hyperreactivity, vocalizations, tremors, convulsions, and seizures. These are collectively referred here as the acute neuronal activation response. Acute neuronal activation is observed in rats, mice, and non-human primates and is quantifiable using a simple scoring system. It is distinct from acute sedation seen with some phosphorothioate-modified antisense oligonucleotides, characterized by loss of spinal reflexes, ataxia, and sedation. The acute neuronal activation response is largely sequence-independent and is driven by ON chelation of divalent cations, particularly influenced by the divalent cations-to-ON ratio in the dosing solution. Acute neuronal activation can be safely mitigated by adjusting this ratio through magnesium supplementation in the ON formulation. We provide a comprehensive framework for quantifying and mitigating the acute neuronal activation response caused by high concentrations of centrally delivered ON therapeutics in preclinical species.

A vector-based strategy for olfactory navigation in Drosophila
Odors serve as essential cues for navigation. Although tracking an odor plume has been modeled as a reflexive process, it remains unclear whether animals can use memories of their past odor encounters to infer the spatial structure of their chemical environment or their location within it. Here we developed a virtual-reality olfactory paradigm that allows head-fixed Drosophila to navigate structured chemical landscapes, offering insight into how memory mechanisms shape their navigational strategies. We found that flies track an appetitive odor corridor by following its boundary, alternating between rapid counterturns to exit the plume and directed returns to its edge. Using a combination of behavioral modeling, functional calcium imaging, and neural perturbations, we demonstrate that this 'edge-tracking' strategy relies on vector-based computations within the Drosophila central complex in which flies store and dynamically update memories of the direction to return them to the plume's boundary. Consistent with this, we find that FC2 neurons within the fan-shaped body, which encode a fly's navigational goal, signal the direction back to the odor boundary when flies are outside the plume. Together, our studies suggest that flies leverage the plume's boundary as a dynamic landmark to guide their navigation, analogous to the memory-based strategies other insects use for long-distance migration or homing to their nests. Plume tracking thus uses components of a conserved navigational toolkit, enabling flies to use memory mechanisms to navigate through a complex shifting chemical landscape.

Selective manipulation of excitatory and inhibitory neurons in top-down and bottom-up visual pathways using ultrasound stimulation
Techniques for precise manipulation of neurons in specific neural pathways are crucial for excitatory/inhibitory (E/I) balance and investigation of complex brain circuits. Low-intensity focused ultrasound stimulation (LIFUS) has emerged as a promising tool for noninvasive deep-brain targeting at high spatial resolution. However, there is a lack of studies that extensively investigate the modulation of top-down and bottom-up corticothalamic circuits via selective manipulation of excitatory and inhibitory neurons. Here, a comprehensive methodology using electrophysiological recording and c-Fos staining is employed to demonstrate pulse repetition frequency (PRF)-dependent E/I selectivity of ultrasound stimulation in the top-down and bottom-up corticothalamic pathways of the visual circuit in rodents. Ultrasound stimulation at various PRFs is applied to either the lateral posterior nucleus of the thalamus (LP) or the primary visual cortex (V1), and multi-channel single-unit activity is recorded from the V1 using a silicon probe. Our results demonstrate that high frequency PRFs, particularly at 3 kHz and 1 kHz, are effective at activating the bidirectional corticothalamic visual pathway. In addition, brain region-specific PRFs modulate E/I cortical signals, corticothalamic projections, and synaptic neurotransmission, which is imperative for circuit-specific applications and behavioral studies.

Microglia Adopt Temporally Specific Subtypes after Irradiation, Correlating with Neuronal Asynchrony
Cranial radiotherapy causes progressive neurocognitive impairments in cancer survivors. Neuroinflammation is believed to be a key contributor, but its dynamics and consequences for brain function remain poorly understood. Here, we performed comprehensive longitudinal profiling, from 6 hours to 1 year after irradiation (IR) of the mouse hippocampus, using transcriptomic, protein and histological analyses. We identified a delayed microglial response coupling interferon signaling to mitotic progression, and a subsequent induction of temporally regulated subtypes. IR rewired the parenchymal phagocyte profiles due to progressive microglial loss, failure of repopulation through self-renewal and compensatory generation of microglia-like cells derived from peripheral monocytes. These findings were confirmed in autopsied human brain. Finally, we demonstrate two phases of neuronal asynchrony, an early one associated with inflammation and a late one associated with synaptic aberrant regulation. These results provide comprehensive, longitudinal insights into microglia responses that can aid tailoring therapies to preserve cognition in cancer survivors.

A Novel Method for Culturing Telencephalic Neurons in Axolotls
The axolotl (Ambystoma mexicanum), a neotenic salamander with remarkable regenerative capabilities, serves as a key model for studying nervous system regeneration. Despite its potential, the cellular and molecular mechanisms underlying this regenerative capacity remain poorly understood, partly due to the lack of reliable in vitro models for axolotl neural cells. In this study, we developed a novel protocol for primary cultures of adult axolotl telencephalon/pallium, enabling the maintenance of viable and functionally active neural cells. Using calcium imaging and immunocytochemistry, we demonstrated the presence of neuronal and glial markers, synaptic connections, and spontaneous calcium activity, highlighting the functional integrity of the cultured cells. Our findings reveal that these cultures can be maintained in both serum and serum-free conditions, with neurons exhibiting robust neurite outgrowth and responsiveness to injury. This protocol addresses a critical gap in axolotl research by providing a controlled in vitro system to study neurogenesis and regeneration. By offering insights into the regenerative mechanisms of axolotl neurons, this work lays the foundation for comparative studies with mammalian systems, potentially informing therapeutic strategies for neurodegenerative diseases and CNS injuries in humans.

Sleep duration and efficiency moderate the effects of prenatal and childhood ambient pollutant exposure on global white matter microstructural integrity in adolescence
Background: Air pollution is a ubiquitous neurotoxicant associated with alterations in structural connectivity. Good habitual sleep may be an important protective lifestyle factor due to its involvement in the brain waste clearance and its bidirectional relationship with immune function. Wearable multisensory devices may provide more objective measures of sleep quantity and quality. We investigated whether sleep duration and efficiency moderated the relationship between prenatal and childhood pollutant exposure and whole-brain white matter microstructural integrity at ages 10-13 years. Methods: We used multi-shell diffusion-weighted imaging data collected on 3T MRI scanners and objective sleep data collected with Fitbit Charge 2 from the 2-year follow-up visit for 2178 subjects in the Adolescent Brain Cognitive Development Study(R). White matter tracts were identified using a probabilistic atlas. Restriction spectrum imaging was performed to extract restricted normalized isotropic (RNI) and directional (RND) signal fraction parameters for all white matter tracts, then averaged to calculate global measures. Sleep duration was calculated by summing the time spent in each sleep stage; sleep efficiency was calculated by dividing sleep duration by time spent in bed. Using an ensemble-based modeling approach, air pollution concentrations of PM2.5, NO2, and O3 were assigned to each child's residential addresses during the prenatal period (9-month average before birthdate) as well as at ages 9-10 years. Multi-pollutant linear mixed effects models assessed the associations between global RNI and RND and sleep-by-pollutant interactions, adjusting for appropriate covariates. Results: Sleep duration interacted with childhood NO2 exposure and sleep efficiency interacted with prenatal O3 exposure to affect RND at ages 10-13 years. Longer sleep duration and higher sleep efficiency in the context of higher pollutant exposure was associated with lower RND compared to those with similar pollutant exposure but shorter sleep duration and lower sleep efficiency. Conclusions: Low-level air pollution poses a risk to brain health in youth, and healthy sleep duration and efficiency may increase resilience to its harmful effects on white matter microstructural integrity. Future studies should evaluate the generalizability of these results in more diverse cohorts as well as utilize longitudinal data to understand how sleep may impact brain health trajectories in the context of pollution over time.

Glutamatergic heterogeneity in the neuropeptide projections from the lateral hypothalamus to the mouse olfactory bulb
The direct pathway from the lateral hypothalamus to the mouse olfactory bulb (OB) includes neurons that express the neuropeptide orexin-A, and others that do not. The OB-projecting neurons that do not express orexin-A are present in an area of the lateral hypothalamus known to contain neurons that express the neuropeptide melanin-concentrating hormone (MCH). We used virally mediated anterograde tract tracing and immunohistochemistry for orexin-A and MCH to demonstrate that the OB is broadly innervated by axon projections from both populations of neurons. Orexin-A and MCH were expressed in each OB layer across its anterior to posterior axis. Both orexin-A and MCH neurons are genetically heterogeneous, with subsets that co-express an isoform of vesicular glutamate transporter (VGLUT). We used high-resolution confocal imaging to test whether the projections from orexin-A and MCH neurons to the OB reflect this glutamatergic heterogeneity. The majority (~57%) of putative orexin-A axon terminals overlapped with VGLUT2, with smaller proportions that co-expressed VGLUT1, or that did not overlap with either VGLUT1 or VGLUT2. In contrast, only ~26% of putative MCH axon terminals overlapped with VGLUT2, with the majority not overlapping with either VGLUT. Therefore, the projections from the lateral hypothalamus to the OB are genetically heterogeneous and include neurons that can release two different neuropeptides. The projections from both populations are themselves genetically heterogeneous with distinct ratios of glutamatergic and non-glutamatergic axon terminals.

Transferrin receptor-binding blood-brain barrier shuttle enhances brain delivery and efficacy of a therapeutic anti-Abeta antibody
Transferrin receptor-1 (TfR1) transcytosis-mediated delivery of therapeutic monoclonal antibodies across the blood-brain barrier (BBB) is a promising concept in drug development for CNS disorders. We sought to investigate brain delivery and efficacy of Aducanumab (Adu), an anti-A{beta} antibody, when fused to a mouse TfR1-binding Fab fragment as BBB shuttle (TfR1-Adu). Automated 3D light sheet fluorescence imaging coupled with computational analysis was applied to evaluate drug IgG distribution and plaque counts throughout the intact brain of transgenic APP/PS1 mice. TfR1-Adu demonstrated enhanced brain delivery and more homogeneous distribution after both acute and chronic dosing in transgenic APP/PS1 mice compared with unmodified Adu. Also, importantly, only unmodified Adu showed perivascular labelling. While high-dose Adu promoted A{beta} plaque depletion in multiple brain regions, similar plaque-clearing efficacy was achieved with a five-fold lower dose of TfR1-Adu. Furthermore, low-dose TfR1-Adu demonstrated greater capacity to reduce congophilic plaque burden. Collectively, these observations strongly support the applicability of TfR1-enabled BBB shuttle strategies to improve brain delivery and plaque-clearing efficacy while mitigating the risk of vascular-associated amyloid-related imaging abnormalities (ARIA) adverse effects associated with current A{beta} immunotherapeutics.

The dynamics of explore-exploit decisions suggest a threshold mechanism for reduced random exploration in older adults
When faced with a choice between exploring an unknown option vs exploiting an option they know well, older adults explore less and exploit more than younger adults. Recent work has suggested that one driver of this age difference in exploration is a reduction in the extent to which older adults use "random exploration" - exploration that is driven by behavioral variability. Here we investigate potential mechanisms for this age-related difference in random exploration through the lens of a drift diffusion model (DDM) of the explore-exploit choice. In this model, random exploration can be modulated by two mechanisms - the fidelity with which information about the choice is represented in the brain, the "signal-to-noise ratio" (SNR), and the amount of information required to make a decision, the "decision threshold." Reduced random exploration in aging could therefore be caused either by an increase in signal-to-noise ratio or an increase in decision threshold in older adults. By fitting the DDM to choices and response times in a sample of healthy younger and older adults, we found that older adults had a lower SNR and a higher threshold than younger adults. We therefore suggest that reduced random exploration in aging is driven by higher response thresholds in older adults which may compensate for the reduced signal-to-noise ratio with which decision information is represented in the brain.

Non-Canonical Role of DNA Mismatch Repair on Sensory Processing in Mice
DNA repair mechanisms are essential for cellular development and function. This is particularly true in post-mitotic neurons, where deficiencies in DNA damage response proteins can result in severe neurodegenerative and neurodevelopmental disorders. One highly conserved factor involved in DNA repair is Mut-S Homolog 2 (Msh2), which is responsible for correcting base-base mismatches and insertion/deletion loops during cell proliferation. However, its role in mature neuronal function remains poorly understood. This study investigates the impact of Msh2 loss on sensory processing in a mouse model. Using electrophysiological and molecular assays, we identified significant deficits in cortical and thalamic sound processing in Msh2-/- mice. These deficits were linked to dysfunction of the thalamic reticular nucleus (TRN), a brain region that critically regulates corticothalamic and thalamocortical activity. Our findings revealed increased oxidative damage, aberrant neuronal activity, and elevated parvalbumin (PV) expression in PV+ interneurons in the TRN of Msh2-/- mice. Additionally, we observed the presence of connexin plaques, indicating that disrupted gap junction formation may contribute to impaired TRN function. These results underscore the critical role of Msh2 in supporting the functionality of PV+ interneurons in the TRN, thereby profoundly influencing sensory processing pathways. This study provides new insights into the importance of DNA repair mechanisms in neuronal development and function, potentially contributing to our understanding of their role in neurological disorders.

3BTRON: A Blood-Brain Barrier Recognition Network
The blood-brain barrier (BBB) plays a crucial role in maintaining brain homeostasis. During ageing, the BBB undergoes structural alterations. Electron microscopy (EM) is the gold standard for studying the structural alterations of the brain vasculature. However, analysis of EM images is time-intensive and can be prone to selection bias, limiting our understanding of the structural effect of ageing on the BBB. Here, we introduce 3BTRON, a deep learning framework for the automated analysis of the BBB architecture (the morphology, structure, and texture of its various components) in EM images. Using age as a readout, we trained and validated our model on a unique dataset (n = 359). We show that the proposed model could confidently identify the BBB architecture of aged mouse brains from young mouse brains across three different brain regions, achieving a sensitivity of 77.8% and specificity of 80.0% post-stratification when predicting on unseen data. Additionally, feature importance methods revealed the spatial features of each image that contributed most to the predictions. These findings demonstrate a new data-driven approach to analysing age-related changes in the architecture of the BBB.

Tiling of large-scaled environments by grid cells requires experience
Grid cells in the medial entorhinal cortex are widely believed to provide a universal spatial metric supporting vector-based navigation irrespective of the spatial scale of an environment. However, using single unit recordings in freely behaving mice, we demonstrate that spatial periodicity in grid cell firing is substantially disrupted when transitioning from a small to a large-scale arena when the scale ratio is larger than the scale ratio of successive grid modules. Remarkably, grid patterns reemerge with experience in the large-scale arena, suggesting that grid cells can learn to represent large-scale spaces with experience.

Rostro-caudal TMS mapping of immediate transcranial evoked potentials reveals a pericentral crescendo-decrescendo pattern
Background: We recently demonstrated that single-pulse TMS of the primary sensorimotor hand area (SM1HAND) elicits an immediate transcranial evoked potential (iTEP). This iTEP response appears within 2-7 ms post-TMS, featuring high-frequency peaks superimposed on a slow positive wave. Here, we used a linear TMS-EEG mapping approach to characterize the rostro-caudal iTEP expression and compared it to that of motor-evoked potentials (MEPs). Methods: In 15 healthy young volunteers (9 females), we identified the iTEP hotspot in left SM1HAND. We applied single biphasic TMS pulses at an intensity of 110% of resting motor threshold over six cortical sites along a rostro-caudal axis (2 cm rostral to 3 cm caudal to the SM1HAND hotspot). We analyzed site-specific iTEP and MEP responses. Results: iTEP magnitude decreased rostrally and caudally from the SM1HAND hotspot. MEPs exhibited a similar rostro-caudal crescendo-decrescendo pattern. While iTEP and MEP response profiles were similar, normalized iTEP amplitudes decayed less rapidly at the first postcentral site. Discussion: These findings support the idea that pericentral iTEPs reflect a direct response signature of the pericentral cortex, possibly involving a synchronized TMS-induced excitation of cortical pyramidal tract neurons. Similar but non-identical rostro-caudal patterns suggest that iTEPs and MEPs may arise from overlapping but distinct neuronal populations.

cxcl18b-defined transitional state-specific nitric oxide drives injury-induced Müller glia cell-cycle re-entry in the zebrafish retina
In lower vertebrates, retinal Muller glia (MG) exhibit a life-long capacity of cell-cycle re-entry to regenerate neurons following the retinal injury. However, the mechanism driving such injury-induced MG cell-cycle re-entry remains incompletely understood. Combining single-cell transcriptomic analysis and in-vivo clonal analysis, we identified previously undescribed cxcl18b-defined MG transitional states as essential routes towards MG proliferation following green/red cone (G/R cone) ablation. Microglial inflammation was necessary for triggering these transitional states, which expressed the gene modules shared by cells of the ciliary marginal zone (CMZ) where life-long adult neurogenesis takes place. Functional studies of the redox properties of these transitional states further demonstrated the regulatory role of nitric oxide (NO) produced by Nos2b in injury-induced MG proliferation. Finally, we developed a viral-based strategy to specifically disrupt nos2b in cxcl18b-defined MG transitional states and revealed the effect of transitional state-specific NO signaling. Our findings elucidate the redox-related mechanism underlying injury-induced MG cell-cycle re-entry, providing insights into species-specific mechanisms for vertebrate retina regeneration.

L-DOPA induces spatially discrete changes in gene expression in the forebrain of mice with a progressive loss of dopaminergic neurons
L-3,4-Dihydroxyphenylalanine (L-DOPA) is effective at alleviating motor impairments in Parkinson's disease (PD) patients but has mixed effects on nonmotor symptoms and causes adverse effects after prolonged treatment. Here, we analyzed the spatial profile of L-DOPA-induced gene expression in the forebrain of mice with an inducible progressive loss of dopaminergic neurons (the TIF-IADATCreERT2 strain), with a focus on the similarities and differences in areas relevant to different PD symptoms. The animals received a 14-day L-DOPA treatment, and 1 h after the final drug injection, a spatial transcriptome analysis was performed on coronal forebrain sections. A total of 121 genes were identified as being regulated by L-DOPA. We found that the treatment had widespread effects extending beyond the primary areas involved in dopamine-dependent movement control. An unsupervised clustering analysis of the transcripts recapitulated the forebrain anatomy and indicated both ubiquitous and region-specific effects on transcription. The changes were most pronounced in layers 2/3 and 5 of the dorsal cortex and the dorsal striatum, where a robust increase in the abundance of activity-regulated transcripts, including Fos, Egr1, and Junb, was observed. Conversely, transcripts with a decreased abundance, e.g., Plekhm2 or Pgs1, were identified primarily in the piriform cortex, the adjacent endopiriform nucleus, and the claustrum. Taken together, our spatial analysis of L-DOPA-induced alterations in gene expression reveals the anatomical complexity of treatment effects, identifying novel genes affected by the drug, as well as molecular activation in brain areas relevant to the nonmotor symptoms of PD.

Investigation of mitochondrial phenotypes in motor neurons derived by direct conversion of fibroblasts from familial ALS subjects
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of motor neurons, leading to fatal muscle paralysis. Familial forms of ALS (fALS) account for approximately 10% of cases and are associated with mutations in numerous genes. Alterations of mitochondrial functions have been proposed to contribute to disease pathogenesis. Here, we employed a direct conversion (DC) technique to generate induced motor neurons (iMN) from skin fibroblasts to investigate mitochondrial phenotypes in a patient-derived disease relevant cell culture system. We converted 7 control fibroblast lines and 17 lines harboring the following fALS mutations, SOD1A4V, TDP-43N352S, FUSR521G, CHCHD10R15L, and C9orf72 repeat expansion. We developed new machine learning approaches to identify iMN, analyze their mitochondrial function, and follow their fate longitudinally. Mitochondrial and energetic abnormalities were observed, but not all fALS iMN lines exhibited the same alterations. SOD1A4V, C9orf72, and TDP-43N352S iMN had increased mitochondrial membrane potential, while in CHCHD10R15L cells membrane potential was decreased. TDP-43N352S iMN displayed changes in mitochondrial morphology and increased motility. SOD1A4V, TDP-43N352S, and CHCHD10R15L iMN had increased oxygen consumption rates and altered extracellular acidification rates, reflecting a hypermetabolic state similar to the one described in sporadic ALS fibroblasts. FUSR521G mutants had decreased ATP/ADP ratio, suggesting impaired energy metabolism. We then tested the viability of iMN and found decreases in survival in SOD1A4V, C9orf72, and FUSR521G, which were corrected by small molecules that target mitochondrial stress. Together, our findings reinforce the role of mitochondrial dysfunction in ALS and indicate that fibroblast-derived iMN may be useful to study fALS metabolic alterations. Strengths of the DC iMN approach include low cost, speed of transformation, and the preservation of epigenetic modifications. However, further refinement of the fibroblasts DC iMN technique is still needed to improve transformation efficiency, reproducibility, the relatively short lifespan of iMN, and the senescence of the parental fibroblasts.