bioRxiv Subject Collection: Neuroscience's Journal

Peripheral anatomy and central connectivity of proprioceptive sensory neurons in the Drosophila wing
Recent advances in electron microscopy (EM) and automated image segmentation have produced synaptic wiring diagrams of the Drosophila central nervous system. A limitation of existing fly connectome datasets is that most sensory neurons are excised during sample preparation, creating a gap between the central and peripheral nervous systems. Here, we bridge this gap by reconstructing wing sensory axons from the Female Adult Nerve Cord (FANC) EM dataset and mapping them to peripheral sensory structures using genetic tools and light microscopy. We confirm the location and identity of known wing mechanosensory neurons and identify previously uncharacterized axons, including a novel population of putative proprioceptors that make monosynaptic connections onto wing steering motor neurons. We also find that proprioceptors of adjacent campaniform sensilla on the wing have distinct axon morphologies and postsynaptic partners, suggesting a high degree of specialization in axon pathfinding and synaptic partner matching. The peripheral location and central projections of wing sensory neurons are stereotyped across flies, allowing this wing proprioceptor atlas and genetic toolkit to guide analysis of other fly connectome datasets.

Uncovering the invisible giant: Amyloid β plaques and their proposed association with waste removal in Alzheimer-affected human hippocampus
According to the prevalent 'Amyloid Hypothesis,' the underlying cause for neurodegeneration in Alzheimer Disease (AD) is attributed to the accumulation of misfolded Amyloid {beta} and tau protein in the form of extracellular sticky plaques and neurofibrillary tangles respectively. These protein accumulations are thought to be caused by impaired waste removal. In an alternative hypothesis, we have proposed the existence of an extensive glial canal system that is likely formed by myelinated aquaporin-4 (AQP4)-expressing tanycytes and removes cellular waste from the hippocampal formation. Here, we demonstrate that tanycyte-derived waste-internalizing receptacles are immunoreactive for A{beta} and emanate from specialized nucleus-like organelles in the following referred to as 'tanysomes.' Utilizing RNA-scope in situ hybridization, we demonstrate that these receptacle-forming tanysomes express RNA for AQP4 and the A{beta}-related genes, amyloid precursor protein, and presenilin 1. These findings suggest that A{beta} is likely synthesized where receptacle formation is observed and that A{beta} may play an important structural role in receptacle formation. In AD-affected hippocampus excessive amounts of A{beta}-immunoreactive waste receptacles emerge from tanysomes and have the appearance of plaques in A{beta}-immunolabeled hippocampus. Moreover, we demonstrate that the same receptacle-forming organelles exhibit strong immunolabeling for hyperphosphorylated tau protein in AD-affected tissue. We postulate that both proteins may play important structural roles in waste uptake and that hypertrophic swelling of impaired tanycytes in AD-affected brain may be due to obstructions of this extensive interconnected glial canal system.

Nucleus accumbens neuron subtype translatome signatures in socially stress females
Background: The nucleus accumbens (NAc) is a reward circuitry hub associated with major depressive disorder (MDD) and chronic stress in rodents. While transcriptional adaptations in NAc medium spiny neuron (MSN) subtypes, those enriched in dopamine receptor 1(D1) or dopamine receptor 2 (D2) and adenosine 2a receptor (A2A) are well characterized in socially stressed male rodents, there is less knowledge of MSN subtype adaptations in socially stressed females. Methods: Chronic witness defeat stress (CWDS) was performed in female D1-Cre-RiboTag and A2A-Cre-RiboTag mice. After stress, mice were separated into high- and low-social interactors with a three Chamber Social Interaction test. Following isolation of ribosome-associated mRNA, MSN subtype-specific gene expression profiles were determined with RNA sequencing followed by differential gene expression analysis (DEG) and weighted gene co-expression network analysis (WGCNA). Consensus WGCNA with male mouse social defeat stress and clinically relevant human transcriptomic datasets was performed to examine the translational sex-specific molecular signatures. Results: 9 DEGs in D1-MSNs and 630 in A2A-MSNs were identified in female stress groups (FDR < 0.05). D1-MSN DEGs were mostly upregulated in CWDS low vs high interactors and were enriched for functions related to energy homeostasis and cell adhesion. A2A-MSN DEGs upregulated in low interactors involved structural molecules while downregulated genes involved neurotransmission. WGCNA identified 9 significant D1- and 5 significant A2A-MSN modules related to cell structure, protein synthesis, synapse and mitochondria. The most impacted modules from each subtype (based on DEG count) were enriched for PI3K-Akt-mTOR signaling pathway and regulated by the Nf1 transcription factor. Consensus module analysis identified a module significantly associated with social stress in a sex- and subtype-specific manner in both mice and humans enriched for genes involved in the PI3K-Akt-mTOR signaling pathway. Conclusion: Chronic social stress-induced sex-specific molecular signatures were uncovered in female subjects. Consensus modules across stress and clinical populations implicate alterations in MSN subtypes could contribute to MDD signatures among female populations.

D1/D5 Receptor Activation Promotes Long-Term Potentiation and Synaptic Tagging/Capture in Hippocampal Area CA2
Hippocampal area CA2 plays an important role in social memory formation. However, CA2 is characterised by plasticity-resistant Schaffer collateral-CA2 (SC-CA2) synapses and highly plastic entorhinal cortex-CA2 (EC-CA2) synapses. Despite abundant dopaminergic input, the relationship between dopamine signalling and area CA2 synaptic plasticity remains unexplored. Here, we show that SKF-38393-mediated Dopamine D1-like receptor (dopamine D1 and D5 receptors) activation differentially primes CA2 inputs in an NMDAR and protein synthesis-dependent manner. We defined an inverted-U shape relationship between SKF-38393 concentration and EC-CA2 potentiation. Additionally, we observed a priming effect on SC-CA2 plasticity with 50M SKF-38393, relieving plasticity resistance. We also demonstrated that this effect follows canonical protein kinase A (PKA) signalling. Collectively, our results show that D1R activation primes the CA2 for synaptic plasticity. Thus, we propose a link between neuropsychiatric diseases related to impaired dopamine transmission and deficits in hippocampus-dependent social memory.

Distributed learning across fast and slow neural systems supports efficient motor adaptation
Adaptation is a fundamental aspect of motor learning. Intelligent systems must adapt to perturbations in the environment while simultaneously maintaining stable memories. Classic work has argued that this trade-off could be resolved by complementary learning systems operating at different speeds; yet the mechanisms enabling coordination between slow and fast systems remain unknown. Here, we propose a multi-region distributed learning model in which learning is shared between two populations of neurons with distinct roles and structures: a recurrent 'controller' network which stores a slowly evolving memory, and a feedforward 'adapter' network that rapidly learns to respond to perturbations in the environment. In our model, supervised learning in the adapter produces a predictive error signal that simultaneously tutors consolidation in the controller through a local plasticity rule. Our model offers insight into the mechanisms that may support distributed computations in the motor cortex and cerebellum during motor adaptation.

Semantic Memory Traces Reflect How They Were Last Retrieved
Episodic memories are known to change with each act of retrieval. We hypothesize that semantic memories are altered in a similar way when retrieval draws on a core dimension of conceptual knowledge: semantic granularity. Each level, from accessing a concept via unique perceptual features to its thematic context, shapes the retrieval route used to access semantic memories. This study tests whether semantic granularities used to retrieve a concept influences its reactivation during later recognition. Human participants learned and retrieved novel word-image pairs while undergoing fMRI. Following encoding, items were retrieved via one of three semantic levels: item, category, or theme. Later, participants accessed the memories in recognition and cued recall tests. Although behavioral memory performance was matched across conditions, neural activity during recognition varied based on prior retrieval history. Recognition patterns could be classified according to prior retrieval granularity in ventral temporal cortex for both remembered and non-remembered concepts, and in the hippocampus for remembered concepts only. A whole-brain searchlight analysis revealed bilateral clusters along the ventral visual stream, from early visual cortex to fusiform gyrus, where retrieval history was decodable. Representational similarity analyses showed that category-level retrieval increased pattern consistency in early visual cortex and pattern reactivation in ventral temporal cortex, while item-level retrieval enhanced memory trace distinctiveness in visual word form area. Theme-level retrieval increased activity in left ventrolateral prefrontal cortex. These findings show that how we access a concept leaves a detectable trace on subsequent neural reactivation, subtly shaping how conceptual knowledge is organized in the brain.

Redundancy masking and the compression of information in the brain
The visual world is inherently complex, presenting far more information than a human visual system can process in full. To manage this overload, the visual brain employs several mechanisms. One mechanism that possibly contributes to the reduction of information is redundancy masking (RM): the reduction of the number of perceived items in repeating patterns. For example, when three identical lines are presented in the periphery, observers often perceive only two. The underlying neural mechanisms of RM remain unclear. Here, we use steady-state visual evoked potential (SSVEP) to examine whether redundancy-masked items are neurally suppressed or integrated with neighboring items. Three identical arcs (quarter-circles; 0.44{degrees} line width) were presented in the periphery (eccentricities: 17.3{degrees}, 19.5{degrees}, and 21.7{degrees}), each tagged with a unique frequency. Participants maintained central fixation, monitored via a gaze-contingent control, and reported the number of arcs they predominantly perceived after each 10s trial. We analyzed baseline-corrected amplitudes at each tagged frequency and calculated signal-to-noise ratios (SNRs) for fundamental and intermodulation (IM) components, separating trials by behavioral responses (RM: 2 items perceived, non-RM: 3 items perceived). Fundamental frequency comparisons revealed that the outer arc elicited higher SSVEP responses than the inner and middle one under RM, with no significant differences between arcs under non-RM. However, fundamental frequency SNRs did not differ between RM and non-RM perceptions. When we compared IM SNRs, the middle and outer arc's combination was significantly higher during RM compared to non-RM, suggesting increased neural integration between them. These results indicate that RM involves a loss of conscious access to visual information, yet corresponding neural signals are not entirely suppressed. Instead, the neural signatures we found suggest the integration with neighboring elements across space and time. We suggest that redundancy-masked items - although unavailable for conscious report- are still observed in the neural signatures of RM.

Shared and distinct microRNA profiles between HT22, N2A and SH-SY5Y cell lines and primary mouse hippocampal neurons
MicroRNAs (miRNA) are small non-coding RNAs that are key negative regulators of gene expression. Their roles include shaping the gene expression landscape during and after brain development by defining and maintaining levels of proteins that generate the distinct morphological and functional properties of neurons and other brain cell types. HT22, N2A, and SH-SY5Y are common immortalized neuronal cell lines that offer simple, less expensive, and time-saving options for in vitro modelling to evaluate miRNA functions. The extent to which these lines reflect primary neurons remains, however, unclear. Here, we benchmarked the miRNA profiles of cultured mouse hippocampal neurons against Argonaute-loaded miRNAs in the adult mouse hippocampus and miRNA data from the hippocampus of patients with drug-resistant temporal lobe epilepsy. We then compared the miRNA expression landscape in HT22, N2A and SH-SY5Y against mouse hippocampal primary cell cultures. We profiled over 700 miRNAs across the lines and detected 310 miRNAs in all four cell types. This included detection of neuron-enriched miRNAs such as miR-124 and miR-128, although the cell lines typically displayed lower levels of these than in primary neurons and reference adult hippocampal tissue. The miRNA profile in the HT22 cell line showed the highest correlation to the mouse primary neuronal cultures. Together, this study provides a dataset on basal miRNA expression across commonly used cell lines for neuroscience research and evidence for both conserved and distinct profiles that should be used to inform decisions on cell lines for modelling brain and miRNA research.

Slow-Timescale Regulation of Dopamine Release and Mating Drive over Days
The rise and fall of motivational states may take place over timescales as long as many days. We used mouse mating behavior to model how the brain orchestrates slow-timescale changes in motivation. Male mice become sexually satiated after successful matings, and their motivation to mate gradually recovers over a week. Using deep-brain fluorescence-lifetime imaging in the medial preoptic area (MPOA), we found that tonic dopamine transmission - which regulates mating drive - also declined after mating and re-emerged over a week. Two mechanisms regulated dopamine transmission. First, successful mating transiently reduced tonic firing of hypothalamic dopamine-releasing neurons, thereby inhibiting dopamine release and mating behavior. Second, mating reduced the ability of these neurons to produce and release dopamine, and this ability gradually returned over the week-long recovery time course. Therefore, fast and slow mechanisms of neuronal plasticity cooperate to control the early and late phases of motivational dynamics, respectively.

Lack of functional STING modulates immunity but does not protect dopaminergic neurons in the alpha-synuclein preformed fibrils Parkinson's Disease mouse model
Microglia response is proposed to be relevant in the neurogenerative process associated with alpha-synuclein (a-syn) pathology in Parkinson's disease (PD). STING is a protein related to the immune sensing of DNA and autophagy, and it has been proposed to be involved in PD neurodegeneration. To investigate this, we injected 10 g of murine pre-formed fibrils (PFFs) of a-syn (or monomeric and PBS as controls) into the striatum of wild-type (WT) and STINGgt/gt mice, which lack functional STING. We examined motor behavior and brain pathology at 1- and 6-months post-injection. STINGgt/gt mice showed more motor changes associated with PFF injection than WT mice. STINGgt/gt mice had a differential immune response to PFF with early and sustained increased microglia numbers and earlier macrophagic CD68 response, but milder changes in the expression of immune-relevant markers such as TLR2, TLR4, IL1b, and TREM2. However, the lack of STING did not induce changes in the extent of a-syn pathology nor the p62 accumulation seen in the model. Altogether, this resulted in a faster but similar degree of nigrostriatal dopaminergic degeneration after 6 months. Therefore, the data do not support a necessary role for STING in the a-syn induced nigral neuronal loss in the PFF-PD mouse model used here. However, the results suggest a functional relevance for STING in the brain response to the excess of amylogenic proteins such as a-syn that can contribute to symptomatic changes.

Stroke-Related Changes in Tonic and Phasic Muscle Recruitment During Reaching Reveal Pathway-Specific Motor Deficits
Background and Purpose: Upper limb motor deficits are common after stroke and often persist despite rehabilitation. Clinical assessments focus on movement quality but fail to quantify underlying neuromuscular impairments, especially in individuals with mild deficits. We aimed to characterize stroke-related changes in muscle recruitment during reaching by separating tonic (gravity-compensating) and phasic (intersegmental dynamics-related) components of electromyographic (EMG) activity and assessing their relationship to motor impairment. Methods: We recorded surface EMG from 12 upper limb muscles during goal-directed reaching in 8 participants with unilateral ischemic stroke and 9 age-matched controls. Using principal component analysis, we extracted tonic and phasic components of muscle activity and compared their amplitude, directional tuning, and coactivation patterns across groups. Score differences between stroke and control groups were analyzed using generalized linear mixed-effects models, regression analyses, and correlation with time since stroke and previously published muscle torque-based performance indices. Results: Individuals with stroke exhibited disrupted muscle recruitment even with mild motor deficits. Proximal muscles were over-recruited in directions that normally require less muscle activation, indicating abnormal directional tuning. Phasic activation of distal muscles was significantly reduced and declined further with time post-stroke (R2 = 0.52, p = 0.002), while tonic overactivation of proximal muscles was present in all participants with stroke. Coactivation between multiple muscles was altered, with right-hemisphere stroke reducing tonic coactivation in contralateral arms and left-hemisphere stroke increasing it. Abnormal phasic coactivation between proximal and distal muscles was present and correlated with impaired limb propulsion (R2 = 0.67, p = 0.013). Tonic and phasic recruitment deficits were often correlated, suggesting shared disruption of corticospinal and reticulospinal pathways. Conclusions: Stroke causes distinct yet interacting impairments in tonic and phasic muscle recruitment that underlie individual motor deficits. These findings support the need for neuromechanically informed assessment tools that target specific motor pathways to guide individualized rehabilitation strategies.

Synthetic Serum Markers Enable Noninvasive Monitoring of Gene Expression in Primate Brains
We demonstrate a noninvasive approach to measure transgene expression in the brain of nonhuman primates using blood tests with engineered reporters called Released Markers of Activity (RMAs). RMAs can exit the brain and enter the blood-stream via reverse-transcytosis across the blood-brain barrier. We demonstrate that these reporters can be used to repeatedly monitor expression of multiple transgenes in cortical and subcortical brain regions simultaneously over a period of two months. RMAs are also sensitive enough to detect circuit-specific Cre-dependent AAV expression. Through this study, the RMA platform provides a cost-efficient, noninvasive tool for neuroscience study of large animals, enabling sensitive, multi-plexed, and repeatable measurements of gene expression in the brain with a blood test.

Block-structured Bayesian source estimation model for magnetoencephalography signals
The human brain consists of functionally specialized areas that process specific types of information and interact with one another. Magnetoencephalography (MEG) is a neuroimaging technique that captures brain activity at high temporal resolution. However, its spatial resolution is insufficient to accurately localize neural activation, and thus cannot capture the interactive nature of human brain activity. To resolve this issue, various MEG source estimation models have been proposed. In particular, models incorporating prior information from functional magnetic resonance imaging (fMRI), which offers superior spatial resolution, have improved source estimation accuracy. However, these models typically ignore the similarity of activity across different brain areas, and still fall short in tracking dynamic brain activity pattern at a sufficient spatial scale. In this study, we developed a block-structured model that integrates information about functional areas and the inter-areal relationships of the human brain into MEG source estimation based on a hierarchical Bayesian framework. We evaluated our model performance using simulation data with sequential activation across multiple brain areas. Results showed that our model outperformed conventional approaches in source estimation accuracy, suggesting that incorporating the functional areal information and inter-areal relationships may enhance MEG source estimation, enabling human neuroimaging at high spatio-temporal resolution.

Spine-Prints: Transposing Brain Fingerprints to the Spinal Cord
Functional connectivity (FC) patterns in the human brain form a reproducible, individual-specific "fingerprint" that allows reliable identification of the same participant across scans acquired over different sessions. While brain fingerprinting is robust across healthy individuals and neuroimaging modalities, little is known about whether the fingerprinting principle extends beyond the brain. Here, we used multiple spinal functional magnetic resonance imaging (fMRI) datasets acquired at different sites to examine whether a fingerprint can be revealed from FCs of the cervical region of the human spinal cord. Our results demonstrate that the functional organisation of the cervical spinal cord also exhibits individual-specific properties, suggesting the potential existence of a spine-print within the same acquisition session. This study provides the first evidence of a spinal cord connectivity fingerprint, underscoring the importance of considering a more comprehensive view of the entire central nervous system. Eventually, these spine-specific signatures could contribute to identifying individualized biomarkers of neuronal connectivity, with potential clinical applications in neurology and neurosurgery.

Visual working memory precision is under voluntary control
The ability to store information in visual working memory is essential to plan and successfully execute memory-guided actions in natural human behavior. Typically, visual working memory research investigates how storing affects subsequent action. In doing so, however, the importance of how the action affects prior storing remains underappreciated. Therefore, we here question how the required precision for an action to succeed, affects how relevant visual information is encoded, maintained and finally acted on. To this end, we had participant memorize 1, 2 or 4 colors for delayed continuous report. Crucially, we manipulated how (im)precise the report was allowed to still be marked correct. Behavioral results showed that for actions with higher required precision, reports became more precise, but only when one or two colors were memorized. Also, reports became slower with higher required precision, regardless of the number of colors. By leveraging pupillometry, we further showed that with higher required precision, 1) colors were encoded deeper (since pupils constricted more during presentation), and 2) more effort was exerted to maintain the colors (since pupils dilated more during retention). Moreover, we found that participants kept exerting more effort to be precise (with increasing precision requirements), even when additional effort did not result in better performance anymore. Our findings demonstrate that humans consider their intended actions when encoding and maintaining information in visual working memory. Our results highlight the essential role of action in understanding how visual information is stored during natural goal-directed behavior.

C9orf72 polyGA knock-in mice exhibit mild motor and proteomic changes consistent with ALS/FTD
A GGGGCC repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The repeat expansion is translated into five different dipeptide repeat proteins: polyGA, polyGP, polyGR, polyAP and polyPR. To investigate the effect of polyGA, which is the most abundant dipeptide repeat protein in patient brains, we used CRISPR/Cas9 to insert 400 codon-optimized polyGA repeats immediately downstream of the mouse C9orf72 start codon. This generated (GA)400 knock-in mice driven by the endogenous mouse C9orf72 promoter, coupled with heterozygous C9orf72 reduction. (GA)400 mice develop subtle pathology including mild motor dysfunction characterized by impaired rotarod performance. Quantitative proteomics revealed polyGA expression caused protein alterations in the spinal cord, including changes in previously identified polyGA interactors. Our findings show that (GA)400 mice are a complementary in vivo model to better understand C9ALS/FTD pathology and determine the specific role of single DPRs in disease.