bioRxiv Subject Collection: Neuroscience's Journal

Altered mechanical properties of astrocytes lacking MLC1; implications for the leukodystrophy MLC
Loss of function of the astrocyte protein MLC1 causes Megalencephalic Leukoencephalopathy with subcortical Cysts (MLC), a leukodystrophy characterized by white matter edema and slow neurological deterioration. MLC1 dysfunction leads to swelling of perivascular astrocyte endfeet and an impaired attachment of endfeet to blood vessels. In isolated primary astrocytes, loss of MLC1 hinders recovery of astrocytes from cell swelling, but the cellular function of MLC1 is not completely understood. MLC1 modulates gating of mechanosensitive ion channels involved in volume regulation. The cytoskeleton plays a crucial role in cell volume regulation, and interactions between the cytoskeleton and cell membrane affect the properties of mechanosensitive ion channels. Therefore, we investigated whether primary Mlc1-null mouse astrocytes show a disruption in their mechanical properties. We measured mechanical properties of cultured primary astrocytes with an indentation technique and demonstrated that Mlc1-null astrocytes are softer than wild-type astrocytes. Proteomic analysis confirmed dysregulation of several cytoskeleton-related pathways in Mlc1-null astrocytes. Confocal imaging revealed that organization of the actin cytoskeleton is unaffected. Instead, we observed alterations in focal adhesions, which aid in relaying mechanical forces between the cytoskeleton, cell membrane, and the extracellular matrix (ECM). Together, our findings reveal that the mechanical properties of Mlc1-null astrocytes are altered, and that disrupted cytoskeleton-membrane-ECM interactions potentially play a role in the disease. Modulators of astrocyte mechanobiology might therefore hold promise for MLC therapy development.

Surface Expansion Regionalization of the Hippocampus in Early Brain Development
The hippocampal formation is implicated in a myriad of crucial functions, particularly centered around memory and emotion, with distinct subdivisions fulfilling specific roles. However, there is no consensus on the spatial organization of these subdivisions, given that the functional connectivity and gene expression-based parcellation along its longitudinal axis differs from the histology-based parcellation along its medial-lateral axis. The dynamic nonuniform surface expansion of the hippocampus during early development reflects the underlying changes of microstructure and functional connectivity, providing important clues on hippocampal subdivisions. Moreover, the thin and convoluted properties bring out the hippocampal maturity largely in the form of expanding surface area. We thus unprecedentedly explore the development-based surface area regionalization and patterns of the hippocampus by leveraging 513 high-quality longitudinal MRI scans during the first two postnatal years. Our findings imply two discrete hippocampal developmental patterns, featuring one pattern of subdivisions along the anterior-posterior axis (head, regions 1 and 5; body, regions 2, 4, 6, and 7; tail, region 3) and the other one along the medial-lateral axis (subiculum, regions 4, 5, and 6; CA fields, regions 1, 2, and 7). Most of the resulting 7 subdivisions exhibit region-specific and nonlinear spatiotemporal surface area expansion patterns with an initial high growth, followed by a transition to low increase. Each subregion displays bilaterally symmetric pattern. The medial portion of the hippocampal head experiences the most rapid surface area expansion. These results provide important references for exploring the fine-grained organization and development of the hippocampus and its intricate cognitions.

Distinct prelimbic cortex ensembles encode response execution and inhibition
Learning when to initiate or withhold actions is essential for survival and requires integration of past experiences with new information to adapt to changing environments. While stable prelimbic cortex (PL) ensembles have been identified during reward learning, it remains unclear how they adapt when contingencies shift. Does the same ensemble adjust its activity to support behavioral suppression upon reward omission, or is a distinct ensemble recruited for this new learning? We used single-cell calcium imaging to longitudinally track PL neurons in rats across operant food reward Training, Extinction and Reinstatement, trained rat-specific decoders to predict trial-wise behavior, and implemented an in-silico deletion approach to characterize ensemble contributions to behavior. We show that operant training and extinction recruit distinct PL ensembles that encode response execution and inhibition, and that both ensembles are re-engaged and maintain their roles during Reinstatement. These findings highlight ensemble-based encoding of multiple learned associations within a region, with selective ensemble recruitment supporting behavioral flexibility under changing contingencies.

The aging human brain exhibits reduced cerebrospinal fluid flow during sleep due to both neural and vascular factors
Aging reduces the quality and quantity of sleep, and greater sleep loss over the lifespan is predictive of neurodegeneration and cognitive decline. One mechanism by which sleep loss could contribute to impaired brain health is through disruption of cerebrospinal fluid (CSF) circulation. CSF is the primary waste transport system of the brain, and in young adults, CSF waves are largest during NREM sleep. However, whether sleep-dependent brain fluid physiology changes in aging is not known, due to the technical challenges of performing neuroimaging studies during sleep. We collected simultaneous fast fMRI and EEG data to measure large-scale CSF flow in healthy young and older adults and tested whether there were age-related changes to CSF dynamics during nighttime sleep. We found that sleep-dependent CSF flow was reduced in older adults, and this reduction was linked to impaired frontal EEG delta power and global hemodynamic oscillations during sleep. To identify mechanisms underlying reduced CSF flow, we used sensory and vasoactive stimuli to drive CSF flow in daytime task experiments, and found that both neural and cerebrovascular physiological changes contributed to the disruption of CSF flow during sleep. Finally, we found that this reduction in CSF flow was associated with gray matter atrophy in aging. Together, these results demonstrate that the aging human brain has reduced CSF flow during sleep, and identifies underlying neurovascular mechanisms that contribute to this age-related decline, suggesting targets for future interventions.

A cortical microcircuit model reveals distinct inhibitory mechanisms of network oscillations and stability
We identify a computational mechanism for network oscillations distinct from classic excitatory-inhibitory networks - CAMINOS (Canonical Microcircuit Network Oscillations) - in which different inhibitory-interneuron classes make distinct causal contributions to network oscillations and stability. A computational network model of the canonical microcircuit consisting of SOM, PV and excitatory neurons reproduced key experimental findings, including: stochastic gamma oscillations with drive-dependent frequency; precise phase-locking of PV interneurons and delayed firing of SOM interneurons; and the distinct effects of optogenetic perturbations of SOM and PV cells. In CAMINOS, the generation of network oscillations depends on both the precise spike timing of SOM and PV interneurons, with PV cells regulating oscillation frequency and network stability, and delayed SOM firing controlling the oscillation amplitude. The asymmetric PV-SOM connectivity is found to be the key source ingredient to generate these oscillations, that naturally establishes distinct PV and SOM-cell spike timing. The CAMINOS model predicts that increased SOM/PV densities along the cortical hierarchy leads to decreased oscillation frequencies (from gamma to alpha/beta) and increased seizure susceptibility, suggesting a unified circuit model for oscillations across different frequency bands.

Demystifying The Myelin g ratio: Its Origin, Derivation and Interpretation
Most studies involving myelin g ratios over the past 120 years assume this metric enumerates changes in myelin thickness (larger g ratio = thinner myelin) with axon or fiber diameter. And, moreover, such changes are directly correlated with internodal function (conduction velocity). However, such assumptions are warranted only in the absence of experimental errors and artifacts (i.e. under theoretical conditions). In reality, g ratios easily under- or overestimate rates of change exceeding 10%, especially for small caliber fibers. Typical analyses of myelin internodes rely on an explicit mathematical model, g ratio=DA/DF, where DA is axon diameter and DF is fiber diameter (myelin plus axon). Shown recently and herein, this model approximates normal physiological conditions only when the axon-fiber diameter relation is directly proportional, whence it is concordant with the axomyelin unit model. However, in transient or non-steady states (development/aging, disease or myelin plasticity) with linear but not directly proportional relations, g ratios poorly describe myelin structure. Acceptance of this counterintuitive assertion is predicated on a detailed understanding of the g ratio - origins, properties and the biology represented - heretofore uncharted. In light of such g ratio limitations, more general and reliable metrics are proposed, the myelin gc ratio and the g` cline.

The neuron-intrinsic membrane skeleton is required for motor neuron integrity throughout lifespan
Axons experience physical stress throughout an organism's lifetime, and disruptions in axonal integrity are hallmarks of both neurodegenerative diseases and traumatic injuries. The spectrin-based membrane periodic skeleton (MPS) is proposed to have a crucial role in maintaining axonal strength, flexibility, and resilience. To investigate the importance of the intrinsic MPS for GABAergic motor neuron integrity in C. elegans, we employed the auxin-inducible degron system to degrade {beta}-spectrin/UNC-70 in a cell-type specific and time-dependent manner. Degradation of {beta}-spectrin from all neurons beginning at larval development resulted in widespread axon breakage and regeneration in VD/DD GABAergic motor neurons in both larval and adult animals. Similarly, targeted degradation of {beta}-spectrin in GABA neurons alone resulted in extensive breakage. Moreover, we found that depleting {beta}-spectrin from the mature nervous system also induced axon breaks. By contrast, epidermal {beta}-spectrin was not required for axon integrity of VD/DD neurons. These findings demonstrate the cell-intrinsic importance of neuronal {beta}-spectrin/UNC-70 for axon integrity both during development and in adulthood.

Long-term development of a motor memory
Human behavior is developed through continuous adaptation to our environment over a range of timescales. Extensive studies have investigated the mechanisms and computations underlying this process of sensorimotor adaptation using several hundred trials. However, most of our motor skills have had countless hours of practice. Here we study a simple motor adaptation task using thousands of training trials over multiple weeks to study the long-term development of a motor memory, and examine changes in adaptation, retention, inter-limb transfer, decay, spontaneous recovery and generalization. Unlike previous studies, participants showed complete compensation to the novel dynamics, along with long-term increases in retention and spontaneous recovery. Moreover, we find narrowing in the angular generalization, suggesting continual tuning of the motor memory to the task. This demonstrates the extensive changes occurring with longer training of motor tasks, highlighting their importance in studies of sensorimotor control, rehabilitation and training.

AFD Thermosensory Neurons Mediate Tactile-Dependent Locomotion Modulation in C. elegans
Sensory neurons drive animal behaviors by detecting environmental stimuli and relaying information to downstream circuits. Beyond their primary roles in sensing, these neurons often form additional synaptic connections outside their main sensory modality, suggesting broader contributions to behavior modulation. Here, we uncover an unexpected role for the thermosensory neuron AFD in coupling tactile experience to locomotion modulation in Caenorhabditis elegans. We show that while AFD employs cGMP signaling for both thermotaxis and tactile-dependent modulation, the specific molecular components of the cGMP pathway differ between these two processes. Interestingly, disrupting the dendritic sensory apparatus of AFD, which is essential for thermotaxis, does not impair tactile-based locomotion modulation, indicating that AFD can mediate tactile-dependent behavior independently of its thermosensory apparatus. In contrast, ablating the AFD neuron eliminates tactile-dependent modulation, pointing to an essential role for AFD itself, rather than its sensory dendritic endings. Further, we find tactile-dependent modulation requires the AIB interneuron, which connects AFD to touch circuits via electrical synapses. Removing innexins expressed in AFD and AIB abolishes this modulation, while re-establishing AFD-AIB connections with engineered electrical synapses restores it. Collectively, these findings uncover a previously unrecognized function of AFD beyond thermosensation, highlighting its influence on context-dependent neuroplasticity and behavioral modulation through broader circuit connectivity.

3D spatial transcriptomics reveals the molecular domain structure of the mouse olfactory bulb
A core organizing principle of the vertebrate brain is its symmetry along multiple axes. However, the structure of these axes, and the precision with which neurons, circuit modules, and brain regions align to them, remain poorly understood. Here, we used 3D spatial transcriptomics to reconstruct the anatomical and molecular organization of the mouse olfactory bulb. We mapped the positions of nearly one thousand molecularly distinct glomeruli, the structural and functional units of odor processing, revealing highly symmetric organization across brain hemispheres. Within each bulb, we defined a curved axis of symmetry that divides pairs of sister glomeruli. Gene expression programs in olfactory sensory neurons predicted glomerular position with near-glomerular resolution. However, glomerular symmetry was disrupted in deeper layer mitral and granule cells, suggesting a reorganization of olfactory bulb output pathways. Our findings provide the first comprehensive map of the molecular domain structure of the olfactory bulb.

EEG responses to auditory cues predict fluency variability and stuttering intervention outcome
Stuttering is a variable speech disorder whose brain mechanisms remain unknown. Sensorimotor brain circuits, critical for motor-speech control, including auditory processing necessary for speech prediction and monitoring, have been linked to the disorder. Despite considerable advances, it remains unclear whether auditory circuits relate to stuttering variability, and whether the panoply of interventions for persons who stutter can lead to brain changes within these circuits. We employed electroencephalography (EEG), in a group of persons who stutter, in combination with auditory probes to tap onto the importance of auditory cortical regions in stuttering variability. Participants produced flexible speech (i.e., describing visual scenes) and non-flexible speech (i.e., reading syllables), following an auditory cue. More pronounced P200 auditory evoked potentials were observed in participants with higher dysfluency rates, mainly in the spontaneous speech task. Interestingly, speech therapy intervention led to a reduction of the P200 potential, which was in turn significantly related to fluency improvements. Furthermore, EEG response patterns discriminative of cue frequency (400 or 800 Hz tones) were also predictive of dysfluency scores. Our study highlights the involvement of auditory cortical processing and that of auditory attention in stuttering variability. We support that a higher state of auditory alertness may be implicated in the sensorimotor mechanisms of stuttering, and that speech therapy interventions promoting more self-confident communication can restraint auditory alertness, and potentially reduce speech dysfluencies.

Quinpirole ameliorates the dysfunction of microglia in human LRRK2-R1441G transgenic mice
Microglia-mediated neuroinflammation is a key contributor to Parkinson' disease (PD) pathogenesis. Leucine-rich repeat kinase 2 (LRRK2), the leading genetic contributor to both familial and sporadic PD, has been implicated in driving this connection. However, its precise role remains incompletely understood due to technical challenges. To address this, we utilized a bacterial artificial chromosome (BAC) transgenic mouse model overexpressing human LRRK2 R1441G, which replicates key features of PD. These mice were crossed with Cx3cr1-EGFP mice to enable assessment of microglial dynamics and function using two-photon imaging in awake mice in vivo and acute brain slices ex vivo. Furthermore, spatial transcriptomic analysis was performed using GeoMx Digital Spatial Profiler technology to compare transgenic mice with their wild-type counterparts. The R1441G mutation upregulated antigen processing and presentation pathways, increased activated microglia, and enhanced microglial polarization in the dorsal striatum. Mutant microglia exhibited reduced motility and slower responses to focal injury, with processes retracting faster and extending more slowly. Quinpirole, a dopamine D2 receptor (D2R) agonist, successfully reversed microglial deficits. This study provides the first evidence that pathogenic LRRK2 mutations alter microglial motility and responsiveness in vivo, highlighting D2R activation as a promising therapeutic strategy to mitigate neuroinflammation and neurodegeneration in PD.

An updated catalogue of split-GAL4 driver lines for descending neurons in Drosophila melanogaster
Descending neurons (DNs) occupy a key position in the sensorimotor hierarchy, conveying signals from the brain to the rest of the body below the neck. In Drosophila melanogaster flies, approximately 480 DN cell types have been described from electron-microscopy image datasets. Genetic access to these cell types is crucial for further investigation of their role in generating behaviour. We previously conducted the first large-scale survey of Drosophila melanogaster DNs, describing 98 unique cell types from light microscopy and generating cell-type-specific split-Gal4 driver lines for 65 of them. Here, we extend our previous work, describing the morphology of 137 additional DN types from light microscopy, bringing the total number DN types identified in light microscopy datasets to 235, or nearly 50%. In addition, we produced 500 new sparse split-Gal4 driver lines and compiled a list of previously published DN lines from the literature for a combined list of 738 split-Gal4 driver lines targeting 171 DN types.

Central amygdalar PKCδ neurons mediate fentanyl withdrawal
Aversion to opioid withdrawal is a significant barrier to achieving lasting opioid abstinence. The central amygdala (CeA), a key brain region for pain, threat-detection, autonomic engagement, and valence assignment, is active during opioid withdrawal. However, the role of molecularly distinct CeA neural populations in withdrawal remains underexplored. Here, we investigated the activity dynamics, brain-wide connectivity, and functional contribution of Protein Kinase C-delta (PKC{delta})-expressing neurons in the CeA lateral capsule (CeLCPKC{delta}) during fentanyl withdrawal in mice. Mapping activity-dependent gene expression in CeLCPKC{delta} neurons revealed a highly withdrawal-active subregion in the anterior half of the CeA. Fiber photometry calcium imaging showed that opioid-naive CeLCPKC{delta} neurons respond to salient noxious and startling stimuli. In fentanyl-dependent mice, naloxone-precipitated withdrawal increased spontaneous neural activity and enhanced responses to noxious stimuli. Chronic inhibition of CeLCPKC{delta} neurons throughout fentanyl exposure, via viral overexpression of the potassium channel Kir2.1, attenuated withdrawal symptoms in fentanyl-dependent mice. Lastly, we identified putative opioid-sensitive inputs to CeLCPKC{delta} neurons using rabies-mediated monosynaptic circuit tracing and color-switching tracers to map mu-opioid receptor-expressing inputs to the CeLC. Collectively, these findings suggest that the hyperactivity of CeLCPKC{delta} neurons underlies the somatic signs of fentanyl withdrawal, offering new insights into the amygdala cell-types and circuits involved in opioid dependence.

Neuronal differentiation enhances a cytoplasmic pool of tousled-like kinase 2 (TLK2)
Mental retardation autosomal dominant 57 (MRD57) is a rare neurodevelopmental disorder characterised by delayed language and psychomotor development, intellectual disability, hypotonia, gastrointestinal issues and facial dysmorphia. It is linked to genetic mutations in the serine/threonine kinase TLK2, characterised by haploinsufficiency and in some cases, its loss or impaired kinase function. TLK2 is an established cell cycle regulator that has been extensively studied in mitotic cells. It is upregulated in cancers, driving tumour growth, however, the role of TLK2 in postmitotic neurons is not understood. We therefore aimed to gain insight into how TLK2 mutations cause MRD57 by determining where TLK2 is expressed in the brain and its subcellular localisation during neuronal differentiation. Public human and mouse brain transcriptomic data revealed splice variant diversity in the N-terminus of TLK2, which contains its nuclear localisation sequence (NLS). Using splice-specific in situ hybridisation probes, we observed expression of TLK2 isoforms that contain and lack the NLS in the mouse hippocampus and cerebellum. We confirmed these findings in human SH-SY5Y neuroblastoma cells, and found that neuronal differentiation of these cells enhances a cytoplasmic pool of TLK2 by two mechanisms: nuclear export of full length TLK2 and increased expression of TLK2 splice variants lacking the NLS. Finally, acute stimuli that mimic synaptic activity were sufficient to elicit nuclear export of TLK2. Our data highlight the need to establish the neuronal cytoplasmic substrates of TLK2 and determine how the loss of TLK2 activity in MRD57 might impact their function in the developing and mature brain.

Synaptotoxic effects of extracellular tau are mediated by its microtubule-binding region
Immunotherapies targeting extracellular tau share the premise that interrupting cell-to-cell spread of tau pathology in Alzheimer's disease (AD) will slow dementia pathogenesis. How these interventions affect the actions of synaptotoxic, extracellular tau species that may help mediate cognitive impairment is relatively unknown. Here, we assayed synaptic plasticity disruption in anaesthetised live rats caused by intracerebral injection of synaptotoxic tau present either in (a) secretomes of induced pluripotent stem cell-derived neurons (iNs) from people with Trisomy 21, the most common genetic cause of AD, or (b) aqueous extracts of human AD brain. Extracellular tau in iN secretomes was found to include fragments that contain the extended microtubule binding regions of tau, MTBR/R' and adjacent C-terminal peptides. Immunodepletion or co-injection with antibodies targeting epitopes within these fragments prevented the acute disruption of synaptic plasticity by these patient-derived synaptotoxic tau preparations. Conversely, a recombinant human tau fragment encompassing the core MTBR/R' - region present in tau fibrils, tau297-391 potently mimicked this deleterious action of patient-derived tau. MTBR/R' -directed antibodies also rapidly reversed a very persistent synaptotoxic effect of soluble brain tau. Our findings reveal a hitherto relatively unexplored potential benefit of targeting MTBR/R'.

Behavioral profiling of hyperbaric oxygen as an intervention for chemotherapy-related functional impairments in male and female mice
Chemobrain or chemotherapy-related cognitive impairment (CRCI) affects up to 75% of cancer patients and survivors following chemotherapy treatments. Chemotherapy typically affects multiple domains including learning, memory, attention, executive function, and mood regulation, persisting for decades after treatment cessation and significantly diminishing cancer survivors quality of life. Despite its prevalence and long-term impact, effective interventions for CRCI remain limited. This study investigated the behavioral effects of HBO on mice exposed to chemotherapy drugs methotrexate (MTX) and 5-fluorouracil (5-FU). Adult male and female C57BL/6 mice received intraperitoneal injections of either saline or chemotherapy (Low-dose: MTX 37.5 mg/kg and 5-FU 50 mg/kg; High-dose: MTX 70 mg/kg and 5-FU 100 mg/kg) once a week for three weeks. Concurrently, subsets of mice underwent daily HBO (2.4 ATA, 90 minutes) five days a week for three weeks. Animals health was evaluated weekly, and behavioral assessment of cognitive, motor, and affective functions was conducted post-treatment. Our results showed that chemotherapy, especially at high-dose, impaired spatial memory and navigation, avoidance learning, fear discrimination, and anxiety regulation differently between males and females. HBO significantly alleviated chemotherapy-induced avoidance learning impairment in both sexes and improved coordinated running capacity in high-dose treated males. However, chemotherapy-HBO cotreatment increased spatial memory deficit in males and increased anxiety-like behaviors in females. In conclusion, even though HBO had some nuanced effects on the various domains, some reversal of CRCI effects were observed. Therefore, HBO should be further studied and considered as a potential treatment for chemobrain.