bioRxiv Subject Collection: Neuroscience's Journal

DMT induces a transient destabilization of whole-brain dynamics
The transition towards the brain state induced by psychedelic drugs is frequently neglected in favor of a static description of their acute effects. We used a time-dependent whole-brain model to reproduce large-scale brain dynamics measured with fMRI from 15 volunteers under 20 mg of bolus intravenous N,N-Dimethyltryptamine (DMT), a short-lasting psychedelic. To capture the transient effects of DMT, we parametrized the proximity to a global bifurcation using a pharmacokinetic equation and adopted the empirical functional connectivity dynamics (FCD) as optimization target. Post-administration, DMT rapidly destabilized brain dynamics, peaking after {approx}5 minutes, followed by a recovery to baseline. Simulated perturbations revealed a transient of heightened reactivity, where stimulation maximally impacted the FCD. Local reactivity concentrated in fronto-parietal regions and visual cortices, and correlated with serotonin 5HT2a receptor density, the primary target of psychedelics. These advances suggest a mechanism to explain key features of the psychedelic state, such as increased brain complexity, diversity, and flexibility. Our model also predicts that the temporal evolution of these features aligns with pharmacokinetics. These results contribute to understanding how psychedelics introduce a transient where minimal perturbations can achieve a maximal effect, shedding light on how short psychedelic episodes may extend an overarching influence over time.

Prestimulus alpha phase modulates visual temporal integration
When presented shortly after another, discrete pictures are naturally perceived as continuous. The neuronal mechanism underlying such continuous or discrete perception are not well understood. While continuous alpha oscillations are a candidate for orchestrating such neuronal mechanisms, recent evidence is mixed. In this study, we investigated the influence of prestimulus alpha oscillation on visual temporal perception. Specifically, we were interested whether prestimulus alpha phase modulates neuronal and perceptual processes underlying discrete or continuous perception. Participant had to report the location of a missing object in a visual temporal integration task, while simultaneously MEG data was recorded. Using source reconstruction, we evaluated local phase effects by contrasting phase angle values between correctly and incorrectly integrated trials. Our results show a phase opposition cluster between - 0.8 to - 0.5 s (relative to stimulus presentation) and between 6 - 20 Hz. These momentary phase angle values were correlated with behavioural performance and event related potential amplitude. There was no evidence that frequency defined a window of temporal integration.

The investigation of dysregulated visual perceptual organization in adults with autism spectrum disorders with phase-amplitude coupling and directed connectivity
Phase-amplitude coupling (PAC) has been used as a powerful tool to understand the mechanism underlying neural binding by investigating neural synchrony across different frequency bands. This study examined the possibility that dysregulated alpha-gamma modulation may be crucially involved in aberrant brain functioning in autism spectrum disorder (ASD). Magnetoencephalographic data were recorded from 13 adult participants with ASD and 16 controls. The time-coursed sources averaged over a primary visual area 1 and fusiform gyrus area were reconstructed with the minimum-norm estimate method. The alpha-gamma PAC was further calculated based on these sources. The statistical analysis was implemented based on the PAC and directed asymmetry index. The results showed the hyper-activity coupling for ASD at the no-face condition and revealed the importance of alpha-gamma phase modulation in detecting a face. Our data provides novel evidence for the role of the alpha-gamma PAC and suggests that the globe connectivity may be more critical during visual perception.

LHX2 regulates dendritic morphogenesis in layer II/III of the neocortex via distinct pathways in progenitors and postmitotic neurons.
In the mammalian neocortex, excitatory neurons that send projections via the corpus callosum are critical to integrating information across the two brain hemispheres. The molecular mechanisms governing the development of the dendritic arbours and spines of these callosal neurons are poorly understood, yet these features are critical to their physiological properties. LIM Homeodomain 2 (Lhx2), a regulator of fundamental processes in cortical development, is expressed in postmitotic callosal neurons occupying layer II/III of the neocortex and also in their progenitors in the embryonic day (E) 15.5 ventricular zone of the mouse neocortex. We tested whether this factor is essential for dendritic arbour configuration and spine morphogenesis of layer II/III neurons. Here, we report loss of Lhx2 either in postmitotic callosal neurons or their progenitors, resulting in shrunken dendritic arbours and perturbed spine morphology. In postmitotic neurons, we identified that LHX2 regulates dendritic and spine morphogenesis via the canonical Wnt/{beta} Catenin signalling pathway. Constitutive activation of this pathway in postmitotic neurons recapitulates the Lhx2 loss-of-function phenotype. In E15.5 progenitors, we identified that bHLH transcription factor Neurog2 mediates LHX2 function in regulating dendritic and spine morphogenesis. We show that Neurog2 expression increases upon loss of Lhx2 and that shRNA-mediated Neurog2 knockdown rescues the loss of Lhx2 phenotype. Our study uncovers novel LHX2 functions in cortical circuit assembly consistent with its temporally dynamic and multifunctional roles in development.

Scale-Dependent Coding of the Hippocampus in Relational Memory
Memory, woven into the very fabric of consciousness, serves as a time-traveling vessel in the mind, using detailed recollections not just for nostalgic reflection but as an abstract map for charting unknown futures. Here, we employed fMRI to investigate how the hippocampus (HPC) encodes detailed experiences into abstract knowledge (i.e., formation) and uses this knowledge for decision making (i.e., utilization) when human participants learned and then utilized spatial-temporal relations in a relational memory task. We found a functional gradient along the anterior-posterior axis of the HPC, characterized by representational similarity and functional connectivity with the autobiographical network. Here, the posterior HPC was more actively engaged in memory formation, whereas the anterior HPC was predominantly involved in memory utilization. Our computational modeling of relational memory further established a causal link between this functional gradient and the HPC's well-documented anatomical gradient, as optimal task performance arose from a combination of a fine-grained representation of past experiences by the posterior HPC and a coarser representation of abstract knowledge for future planning by the anterior HPC. This scale-dependent coding scheme led to the emergence of grid-like, heading direction-like, and place-like units in our neural network model, analogous to those discovered in biological brains. Taken together, our study revealed the HPC's functional gradient in representing relational memory, and further connected it to the anatomical gradient of place cells, supporting a unified framework where both spatial and episodic memory rely on relational representations that integrate spatial localization with temporal continuity.

Dysregulated acetylcholine-mediated dopamine neurotransmission in the eIF4E Tg mouse model of autism spectrum disorders.
Autism Spectrum Disorders (ASD) consist of diverse neurodevelopmental conditions where core behavioral symptoms are critical for diagnosis. Altered dopamine neurotransmission in the striatum has been suggested to contribute to the behavioral features of ASD. Here, we examine dopamine neurotransmission in a mouse model of ASD characterized by elevated expression of the eukaryotic initiation factor 4E (eIF4E), a key regulator of cap-dependent translation, using a comprehensive approach that encompasses genetics, behavior, synaptic physiology, and imaging. The results indicate that increased eIF4E expression leads to behavioral inflexibility and impaired striatal dopamine release. The loss of normal dopamine neurotransmission is due to a defective nicotinic receptor signaling that regulates calcium dynamics in dopaminergic axons. These findings reveal an intricate interplay between eIF4E, DA neurotransmission, and behavioral flexibility, provide a mechanistic understanding of ASD symptoms and offer a foundation for targeted therapeutic interventions.

Specific amygdala and hippocampal subfield volumes in social anxiety disorder and their relation to clinical characteristics; an international mega-analysis
Background: Social anxiety disorder (SAD) has been associated with alterations in amygdala and hippocampal volume but there is mixed evidence for the direction of volumetric alterations. Additionally, little is known about the involvement distinct subfields of these regions in the pathophysiology of SAD. Methods: T1-weighted MRI images from a large multi-centre sample of 107 adult patients with SAD and 140 healthy controls (HCs) were segmented using FreeSurfer to produce 9 amygdala and 12 hippocampal subfield volumes. Volumes were compared between groups using linear mixed-effects models adjusted for age, age-squared, sex, site and whole amygdala and hippocampal volumes. Subgroup analyses examined subfield volumes in relation to comorbid anxiety disorder, and comorbid major depressive disorder (MDD), psychotropic medication status, and symptom severity. Results: SAD was associated with smaller amygdala volumes in the basal (d=-0.32, pFDR=0.022), accessory basal (d=-0.42, pFDR=0.005) and corticoamygdaloid transition area (d=-0.37, pFDR=0.014), and larger hippocampal volume in the CA3 (d=0.34, pFDR=0.024), CA4 (d=0.44, pFDR=0.007), dentate gyrus (d=0.35, pFDR=0.022) and molecular layer (d=0.28, pFDR= 0.033), compared to HCs. Patients with SAD without comorbid anxiety, in addition, had smaller lateral amygdala (d=-0.30, pFDR=0.037) and hippocampal amygdala transition area (d=-0.33, pFDR=0.027) compared to HCs. In patients with SAD without comorbid MDD, only the smaller accessory basal amygdala remained significant (d=-0.41, pFDR=0.017). No association was found between subfield volume and medication status or symptom severity. Conclusions. We observed distinct patterns of volumetric differences across specific amygdala and hippocampal subfields, regions that are associated with sensory information processing, threat evaluation and fear generalization. These findings suggest a possible disruption in information flow between the amygdala and hippocampal formation for fear processing in SAD.

Cellular Modeling of CLN6 with IPSC-derived Neurons and Glia
Neuronal ceroid lipofuscinosis (NCL), type 6 (CLN6) is a neurodegenerative disorder associated with progressive neurodegeneration leading to dementia, seizures, and retinopathy. CLN6 encodes a resident-ER protein involved in trafficking lysosomal proteins to the Golgi. CLN6p deficiency results in lysosomal dysfunction and deposition of storage material comprised of Nile Red+ lipids/proteolipids that include subunit C of the mitochondrial ATP synthase (SUBC). White matter involvement has been recently noted in several CLN6 animal models and several CLN6 subjects had neuroimaging was consistent with leukodystrophy. CLN6 patient-derived induced pluripotent stem cells (IPSCs) were generated from several of these subjects. IPSCs were differentiated into oligodendroglia or neurons using well-established small-molecule protocols. A doxycycline-inducible transgenic system expressing neurogenin-2 (the I3N-system) was also used to generate clonal IPSC-lines (I3N-IPSCs) that could be rapidly differentiated into neurons (I3N-neurons). All CLN6 IPSC-derived neural cell lines developed significant storage material, CLN6-I3N-neuron lines revealed significant Nile Red+ and SUBC+ storage within three and seven days of neuronal induction, respectively. CLN6-I3N-neurons had decreased tripeptidyl peptidase-1 activity, increased Golgi area, along with increased LAMP1+ in cell bodies and neurites. SUBC+ signal co-localized with LAMP1+ signal. Bulk-transcriptomic evaluation of control- and CLN6-I3N-neurons identified >1300 differentially-expressed genes (DEGs) with Gene Ontogeny (GO) Enrichment and Canonical Pathway Analyses having significant changes in lysosomal, axonal, synaptic, and neuronal-apoptotic gene pathways. These findings indicate that CLN6-IPSCs and CLN6-I3N-IPSCs are appropriate cellular models for this disorder. These I3N-neuron models may be particularly valuable for developing therapeutic interventions with high-throughput drug screening assays and/or gene therapy.

Creating something out of nothing: Symbolic and non-symbolic representations of numerical zero in the human brain
Representing the quantity zero is considered a unique achievement of abstract human thought. Despite considerable progress in understanding the neural code supporting natural numbers, how numerical zero is encoded in the human brain remains unknown. We find that both non-symbolic empty sets (the absence of dots on a screen) and symbolic zero ("0") occupy ordinal positions along graded neural number lines within posterior association cortex. Neural representations of zero are partly independent of numerical format, exhibiting distance effects with countable numerosities in the opposing (symbolic or non-symbolic) notation. Our results show that format-invariant neural magnitude codes extend to judgements of numerical zero, and offer support to theoretical accounts in which representations of symbolic zero are grounded in more basic representations of sensory absences.

Modulation of locomotion and motor neuron response by the cohesive effect of acute and chronic feeding states and acute d-amphetamine treatment in zebrafish larvae.
Amphetamine (AMPH) increases locomotor activities in animals, and the locomotor response to AMPH is further modulated by caloric deficits such as food deprivation and restriction. The increment in locomotor activity regulated by AMPH-caloric deficit concomitance can be further modulated by varying feeding schedules (e.g. acute and chronic food deprivation and acute feeding after chronic food deprivation). However, the effects of different feeding schedules on AMPH-induced locomotor activity are yet to be explicated. Here, we have explored the stimulatory responses of acutely administered d-amphetamine in locomotion under systematically varying feeding states (fed/sated and food deprivation) and schedules (chronic and acute) in zebrafish larvae. We used wild-type and transgenic [Tg(mnx1:GCaMP5)] zebrafish larvae and measured swimming activity and spinal motor neuron activity in vivo in real-time in time-elapsed and cumulative manner pre- and post-AMPH treatment. Our results showed that locomotion and motor neuron activity increased in both chronic and acute food deprivation post-AMPH treatment cumulatively. A steady increase in locomotion was observed in acute food-deprivation compared to an immediate abrupt increase in chronic food-deprivation state. The ad libitum-fed larvae exhibited a moderate increase both in locomotion and motor neuron activity. Conversely to all other caloric states, food-sated (acute feeding after chronic food deprivation) larvae moved moderately less and exhibited a mild decrease in motor neuron activity after AMPH treatment. These results point to the importance of the feeding schedule in modulating characteristic stimulatory response of amphetamine on behavior and motor neurons.

The CD74 inhibitor DRhQ improves cognition and mitochondrial function in 5xFAD mouse model of Aβ accumulation
Neuroinflammation and mitochondrial dysfunction are early events in Alzheimer's disease (AD) and contribute to neurodegeneration and cognitive impairment. Evidence suggests that the inflammatory axis mediated by macrophage migration inhibitory factory (MIF) binding to its receptor, CD74, plays an important role in many central nervous system (CNS) disorders like AD. Our group has developed DRhQ, a novel CD74 binding construct that competitively inhibits MIF binding, blocks T-cell and macrophage activation and migration into the CNS, enhances anti-inflammatory microglia cell numbers and reduces proinflammatory gene expression. Here we evaluate its effects in beta-amyloid (Abeta) overexpressing mice. 5xFAD mice and their wild type littermates were treated with DRhQ (100 ug) or vehicle for 4 weeks. DRhQ improved cognition and cortical mitochondrial function in both male and female 5xFAD mice. Abeta plaque burden in 5xFAD animals were not robustly impacted by DRhQ treatment nor was microglial activation, although in the hippocampus there was some evidence of a reduction in female 5xFAD mice. Future studies are needed to confirm this possible sex-dependent response on microglial activation as well as to optimize the dose, and timing of DRhQ treatment and gain a better understanding of its mechanism of action.

Proximity labeling proteomics reveals Kv1.3 potassium channel immune interactors in microglia.
Microglia are the resident immune cells of the brain and regulate the brain's inflammatory state. In neurodegenerative diseases, microglia transition from a homeostatic state to a state referred to as disease associated microglia (DAM). DAM express higher levels of proinflammatory signaling, like STAT1 and TLR2, and show transitions in mitochondrial activity toward a more glycolytic response. Inhibition of Kv1.3 decreases the proinflammatory signature of DAM, though how Kv1.3 influences the response is unknown. Our goal was to establish the potential proteins interacting with Kv1.3 during the TLR4-mendiated transition to DAM. We utilized TurboID, a biotin ligase, fused to Kv1.3 to evaluate the potential interacting proteins with Kv1.3 via mass spectrometry in BV-2 microglia during an immune response. Electrophysiology, western blots, and flow cytometry were used to evaluate Kv1.3 channel presence and TurboID biotinylation activity. We hypothesized that Kv1.3 contains domain-specific interactors that vary during an TLR4-induced inflammatory response, some of which are dependent on the PDZ-binding domain on the C-terminus. We determined that the N-terminus of Kv1.3 is responsible for trafficking Kv1.3 to the cell surface and mitochondria (i.e. NUNDC, TIMM50). The C-terminus interacts with immune signaling proteins in an LPS-induced inflammatory response (i.e. STAT1, TLR2, and C3). There are 70 proteins that rely on the c-terminal PDZ-binding domain to interact with Kv1.3 (i.e. ND3, Snx3, and Sun1). Overall, we highlight that the Kv1.3 potassium channel functions beyond outward flux of potassium in an inflammatory context and contributes to activity of key immune signaling proteins, such as STAT1 and C3.

Non-CG DNA methylation and MeCP2 stabilize repeated tuning of long genes that distinguish closely related neuron types
The extraordinary diversity of neuron types in the mammalian brain is delineated at the highest resolution by subtle gene expression differences that may require specialized molecular mechanisms to be maintained. Neurons uniquely express the longest genes in the genome and utilize neuron-enriched non-CG DNA methylation (mCA) together with the Rett syndrome protein, MeCP2, to control gene expression, but the function of these unique gene structures and machinery in regulating finely resolved neuron type-specific gene programs has not been explored. Here, we employ epigenomic and spatial transcriptomic analyses to discover a major role for mCA and MeCP2 in maintaining neuron type-specific gene programs at the finest scale of cellular resolution. We uncover differential susceptibility to MeCP2 loss in neuronal populations depending on global mCA levels and dissect methylation patterns and intragenic enhancer repression that drive overlapping and distinct gene regulation between neuron types. Strikingly, we show that mCA and MeCP2 regulate genes that are repeatedly tuned to differentiate neuron types at the highest cellular resolution, including spatially resolved, vision-dependent gene programs in the visual cortex. These repeatedly tuned genes display genomic characteristics, including long length, numerous intragenic enhancers, and enrichment for mCA, that predispose them to regulation by MeCP2. Thus, long gene regulation by the MeCP2 pathway maintains differential gene expression between closely-related neurons to facilitate the exceptional cellular diversity in the complex mammalian brain.

Spike transmission failures in axons from mouse cortical pyramidal neurons in vivo
The propagation of action potentials along axons is traditionally considered to be reliable, as a consequence of the high safety factor of action potential propagation. However, numerical simulations have suggested that, at high frequencies, spikes could fail to invade distal axonal branches. Given the complex morphologies of axonal trees, with extensive branching and long-distance projections, spike propagation failures could be functionally important. To explore this experimentally in vivo, we used an axonal-targeted calcium indicator to image action potentials at axonal terminal branches in superficial layers from mouse somatosensory cortical pyramidal neurons. We activated axons with an extracellular electrode, varying stimulation frequencies, and computationally extracted axonal morphologies and associated calcium responses. We find that axonal boutons have higher calcium accumulations than their parent axons, as was reported in vitro. But, contrary to previous in vitro results, our data reveal spike failures in a significant subset of branches, as a function of branching geometry and spike frequency. The filtering is correlated with the geometric ratio of the branch diameters, as expected by cable theory. These findings suggest that axonal morphologies contribute to signal processing in the cortex.

Adult sex change leads to extensive forebrain reorganization in clownfish
Sexual differentiation of the brain occurs in all major vertebrate lineages but is not well understood at a molecular and cellular level. Unlike most vertebrates, sex-changing fishes have the remarkable ability to change reproductive sex during adulthood in response to social stimuli, offering a unique opportunity to understand mechanisms by which the nervous system can initiate and coordinate sexual differentiation. This study explores sexual differentiation of the forebrain using single nucleus RNA-sequencing in the anemonefish Amphiprion ocellaris, producing the first cellular atlas of a sex-changing brain. We uncover extensive sex differences in cell type-specific gene expression, relative proportions of cells, baseline neuronal excitation, and predicted inter-neuronal communication. Additionally, we identify the cholecystokinin, galanin, and estrogen systems as central molecular axes of sexual differentiation. Supported by these findings, we propose a model of neurosexual differentiation in the conserved vertebrate social decision-making network spanning multiple subtypes of neurons and glia, including neuronal subpopulations within the preoptic area that are positioned to regulate gonadal differentiation. This work deepens our understanding of sexual differentiation in the vertebrate brain and defines a rich suite of molecular and cellular pathways that differentiate during adult sex change in anemonefish.

Cisplatin drives mitochondrial dysregulation in sensory hair cells
Cisplatin is a commonly used chemotherapy that causes permanent hearing loss by injuring cochlear hair cells. The underlying mechanisms that drive hair cell loss remain unknown, but mitochondria have emerged as potential mediators of cisplatin ototoxicity. Direct observation of changes in hair cell mitochondrial function are challenging because the mammalian inner ear is optically inaccessible. Here, we perform live in vivo imaging of hair cells within the zebrafish lateral-line organ to evaluate the role of mitochondria in cisplatin ototoxicity. Using a genetically encoded biosensor that measures cumulative mitochondrial activity in hair cells, we demonstrate that greater redox history increases susceptibility to cisplatin. Next, we conduct time-lapse imaging of individual hair cells to understand dynamic changes in mitochondrial homeostasis. We observe spikes in mitochondrial calcium and cytosolic calcium immediately prior to hair cell death. Furthermore, we use a mitochondrially-localized probe that fluoresces in the presence of cisplatin to show that cisplatin accumulates in hair cell mitochondria. Lastly, we demonstrate that this accumulation occurs before mitochondrial dysregulation, Caspase-3 activation, and ultimately, hair cell death. Our findings provide additional evidence that suggest mitochondria are integral to cisplatin ototoxicity and cisplatin directly targets hair cell mitochondria.

Nutrient Abundance Signals the Changing of the Seasons by Phosphorylating PER2
The circadian clock synchronizes metabolic and behavioral cycles with the rotation of the Earth by integrating environmental cues, such as light. Nutrient content also regulates the clock, though how and why this environmental signal affects the clock remains incompletely understood. Here, we elucidate a role for nutrient in regulating circadian alignment to seasonal photoperiods. High fat diet (HFD) promoted entrainment to a summer light cycle and inhibited entrainment to a winter light cycle by phosphorylating PER2 on serine 662. PER2-S662 phospho-mimetic mutant mice were incapable of entraining to a winter photoperiod, while PER2-S662 phospho-null mutant mice were incapable of entraining to a summer photoperiod, even in the presence of HFD. Multi-omic experimentation in conjunction with isocaloric hydrogenated-fat feeding, revealed a role for polyunsaturated fatty acids in nutrient-dependent seasonal entrainment. Altogether, we identify the mechanism whereby nutrient content shifts circadian rhythms to anticipate seasonal photoperiods in which that nutrient state predominates.

Adapting and optimizing GCaMP8f for use in C. elegans
Improved genetically-encoded calcium indicators (GECIs) are essential for capturing intracellular dynamics of both muscle and neurons. A novel set of GECIs with ultra-fast kinetics and high sensitivity was recently reported by Zhang et al. (Nature, 2023). While these indicators, called jGCaMP8, were demonstrated to work in Drosophila and mice, data for Caenorhabditis elegans was not reported. Here, we present an optimized plasmid for C. elegans and use this to generate several strains expressing GCaMP8f. Utilizing the myo-2 promoter, we compare pharyngeal muscle activity measured with GCaMP7f and GCaMP8f and find that GCaMP8f is brighter, shows faster kinetics and is less disruptive to the intrinsic contraction dynamics of the pharynx. Additionally, we validate its application for detecting neuronal activity in touch receptor neurons which reveals robust calcium transients at 25 ms time resolution . As such, we establish GCaMP8f as a potent tool for C. elegans research which is capable of extracting fast calcium dynamics at very low magnifications across multiple cell types.