bioRxiv Subject Collection: Neuroscience's Journal

Three-photon in vivo imaging of neurons and glia in the medial prefrontal cortex with sub-cellular resolution
The medial prefrontal cortex (mPFC) is important for higher cognitive functions, including working memory, decision making, and emotional control. In vivo recordings of neuronal activity in the mPFC have been achieved via invasive electrical and optical approaches. Here we apply low invasive three-photon in vivo imaging in the mPFC of the mouse at unprecedented depth. Specifically, we measure neuronal and astrocytic Ca2+-transient parameters in awake head-fixed mice up to a depth of 1700 micrometer. Furthermore, we longitudinally record dendritic spine density (0.41 +/-0.07 per micrometer) deeper than 1 mm for a week. Using 1650 nm wavelength to excite red fluorescent microglia, we quantify their processes motility (58.9 +/-2% turnover rate) at previously unreachable depths (1100 micrometer). We establish three-photon imaging of the mPFC enabling neuronal and glial recordings with subcellular resolution that will pave the way for novel discoveries in this brain region.

The Human Cerebellum: A Digital Anatomical Atlas at the Level of Individual Folia
Scientific interest in the cerebellum has surged in the last few decades with an emerging consensus on a multifaceted functionality and intricate, but not yet fully understood, functional topography over the cerebellar cortex. To further refine this structure-function relationship and quantify its inter-subject variability, a high-resolution digital anatomical atlas is fundamental. Using a combination of manual labeling and image processing, we turned a recently published reconstruction of the human cerebellum, the first such reconstruction fine enough to resolve the individual folia, into a digital atlas with both surface and volumetric representations. Its unprecedented granularity (0.16 mm) and detailed expert labeling make the atlas usable as an anatomical ground truth, enabling new ways of analyzing and visualizing cerebellar data through its digital format.

Flanker task parameters are related to the strength of association between the ERN and anxiety: a meta-analysis
The error-related negativity (ERN), an index of error monitoring, is associated with anxiety symptomatology. Although recent work suggests associations between the ERN and anxiety are relatively modest, little attention has been paid to how variation in task parameters may influence the strength of ERN-anxiety associations. To close this gap, the current meta-analysis assesses the possible influence of task parameter variation in the Flanker task, the most commonly used task to elicit the ERN, on observed ERN-anxiety associations. Here, we leveraged an existing open database of published/unpublished ERN-anxiety effect sizes, supplementing this database by further coding for variation in stimulus type (letter vs. arrow), response type (one-handed vs. two-handed), and block-level feedback (with vs. without). We then performed meta-regression analyses to assess whether variation in these Flanker task parameters moderated the effect size of ERN-anxiety associations. No evidence for an effect of stimulus type was identified. However, both response type and block-level feedback significantly moderated the magnitude of ERN-anxiety associations. Specifically, studies employing either a two-handed (vs. one-handed) task, or those with (vs. without) block-level feedback exhibited more than a two-fold increase in the estimated ERN-anxiety effect size. Thus, accounting for common variation in task parameters may at least partially explain apparent inconsistencies in the literature regarding the magnitude of ERN-anxiety associations. At a practical level, these data can inform the design of studies seeking to maximize ERN-anxiety associations. At a theoretical level, the results also inform testable hypotheses regarding the exact nature of the association between the ERN and anxiety.

Effects of connectivity hyperalignment (CHA) on estimated brain network properties: from coarse-scale to fine-scale
Recent gains in functional magnetic resonance imaging (fMRI) studies have been driven by increasingly sophisticated statistical and computational techniques and the ability to capture brain data at finer spatial and temporal resolution. These advances allow researchers to develop population-level models of the functional brain representations underlying behavior, performance, clinical status, and prognosis. However, even following conventional preprocessing pipelines, considerable inter-individual disparities in functional localization persist, posing a hurdle to performing compelling population-level inference. Persistent misalignment in functional topography after registration and spatial normalization will reduce power in developing predictive models and biomarkers, reduce the specificity of estimated brain responses and patterns, and provide misleading results on local neural representations and individual differences. This study aims to determine how connectivity hyperalignment (CHA), an analytic approach for handling functional misalignment, can change estimated functional brain network topologies at various spatial scales from the coarsest set of parcels down to the vertex-level scale. The findings highlight the role of CHA in improving inter-subject similarities, while retaining individual-specific information and idiosyncrasies at finer spatial granularities. This highlights the potential for fine-grained connectivity analysis using this approach to reveal previously unexplored facets of brain structure and function.

Information-making processes in the speaker's brain drive human conversations forward
A conversation following an overly predictable pattern is likely boring and uninformative; conversely, if it lacks structure, it is likely nonsensical. The delicate balance between predictability and surprise has been well studied using information theory during speech perception, focusing on how listeners predict upcoming words based on context and respond to unexpected information. However, less is known about how speakers' brains generate structured yet surprisingly informative speech. This study uses continuous electrocorticography (ECoG) recordings during free, 24/7 conversations to investigate the neural basis of speech production and comprehension. We employed large language models (Llama-2 and GPT-2) to calculate word probabilities based on context and categorized words into probable (top 30%) and improbable (bottom 30%) groups. We then extracted word embeddings from the LLMs and used encoding models to estimate the neural activity while producing or listening to probable and improbable words. Our findings indicate that before word-onset, the human brain functions in opposing, perhaps complementary, ways while listening and speaking. Results show that listeners exhibit increased neural encoding for predictable words before word onset, while speakers show increased encoding for surprising, improbable words. Speakers also show a lower speech production rate before articulating unexpected words, suggesting additional cognitive processes are involved in producing novel information. This indicates that human speech production includes information-making processes for generating informative words that are absent in language models, which primarily rely on statistical probabilities to generate contextually appropriate speech.

CDKL5's role in microtubule-based transport and cognitive function
Cyclin-dependent kinase like 5 (CDKL5) is a serine-threonine kinase highly enriched in mammalian neurons. CDKL5 is located on the X-chromosome and its loss-of-function leads to a severe neurodevelopmental disorder called CDKL5 deficiency disorder (CDD). CDKL5 phosphorylates microtubule-associated protein MAP1S and regulates its binding to microtubules. How MAP1S phosphorylation affects microtubule function is not well understood. To address this question, we generated MAP1S phosphomutant mice, in which the CDKL5 phosphorylation sites S786 and S812 are mutated to Alanine (MAP1S S786/812A or MAP1S SA). Using a microtubule co-sedimentation assay, we showed that dynein binding to microtubules is severely reduced in CDKL5 knockout (KO) and MAP1S SA brains. Time-lapse imaging in primary neurons showed impaired dynein motility in both Cdkl5 KO and MAP1S SA. Dynein-driven cargo transport was affected in mutant neuron dendrites, including the delivery of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. We next studied tubulin tyrosination in Cdkl5 KO and MAP1S SA neurons and found that both mutants had a reduced tubulin tyrosination when compared to WT neurons. Since dynein-dynactin has a higher affinity for tyrosinated microtubules, we hypothesized that reduced tyrosination in MAP1S phosphomutant mice could be the mechanistic cause of impaired dynein motility. In support of this, we show that upon expression of tubulin tyrosine kinase TTL, we rescued dynein motility defects in MAP1S phosphomutant neurons. Hippocampal neurons derived from MAP1S SA mice revealed a significant reduction in spine density and synapses, and altered spine morphology. Finally, behavioral phenotyping of MAP1S phosphomutant mice showed increased anxiety, impaired motor performance, social and memory deficits, mirroring to some extent the clinical manifestations present in CDD patients. Our results reveal MAP1S phosphorylation to be an important contributor to dynein-mediated transport and synapse formation.

Postural demands modulate tactile perception in the lower limb in young and older adults
Balance control requires constant integration of feedforward and feedback signals. In healthy aging, the quality of feedback signals decreases while feedforward control is upweighted; but it is unclear how tactile perception is modulated when balance control is challenged and how this interacts with age-related changes in sensorimotor processes. We therefore examined tactile perception in standing when confronted with different postural demands in young and older adults. To this end, we measured tactile sensitivity on the calf during sitting (baseline), standing on solid ground, and standing on unstable ground (foam). We also measured the center of pressure during standing using a force plate and calculated a 95% confidence ellipse area and the center of pressure length. Tactile sensitivity was assessed by fitting a psychometric function to verbal responses for detecting vibrotactile probes, calculating the detection threshold at 50% detection, and normalizing the two standing conditions to baseline. We examined the effect of age and postural demands on the center of pressure kinematics and detection thresholds. We found higher sway and poorer tactile sensitivity when standing on foam irrespective of age. The increase of postural demands seems to reduce the reliance on tactile feedback signals from the lower limbs in both young and older adults. Our results suggest that postural demands challenge healthy agers as young adults, probably leading to a down-weighting of tactile feedback processing.

Does slow oscillation-spindle coupling contribute to sleep-dependent memory consolidation? A Bayesian meta-analysis
The active system consolidation theory suggests that information transfer between the hippocampus and cortex during sleep underlies memory consolidation. Neural oscillations during sleep, including the temporal coupling between slow oscillations (SO) and sleep spindles (SP), may play a mechanistic role in memory consolidation. However, differences in analytical approaches and the presence of physiological and behavioral moderators have led to inconsistent conclusions. This meta-analysis, comprising 23 studies and 297 effect sizes, focused on four standard phase-amplitude coupling measures including coupling phase, strength, percentage, and SP amplitude, and their relationship with memory retention. We developed a standardized approach to incorporate non-normal circular- linear correlations. We found strong evidence supporting that precise and strong SO-fast SP coupling in the frontal lobe predicts memory consolidation. The strength of this association is mediated by memory type, aging, and dynamic spatio-temporal features, including SP frequency and cortical topography. In conclusion, SO-SP coupling should be considered as a general physiological mechanism for memory consolidation.

How Alzheimer's Abeta propagates and triggers tau pathology in intact neurons
Alzheimer's disease begins with Abeta accumulation, progresses to tau aggregates and results in widespread neurodegeneration (1-3). Simultaneous propagation of Abeta aggregates from very limited to wide and distant brain regions is one of the outstanding events in the early stage. Here, we demonstrate that the neurovascular unit (4,5) comprising capillaries and pericytes, the machinery supplying oxygen and glucose to neurons, is also the machinery for propagating effector molecules that impose Alzheimer's pathologies on intact neurons. We discovered two distinct signaling cascades, one activated in capillary endothelial cells and the other in pericytes. At the origin of either cascade, we identified amylospheroid (ASPD) (6,7), a highly toxic 30-mer assembly of Abeta, and its sole target NKAalpha3 (8), a neuron-specific isoform of Na+,K+-ATPase (9,10) but present in endothelial cells and pericytes. In endothelial cells, ASPD binding to NKAalpha3 releases angiotensin II, which increases beta-secretase in intact neurons, causing a huge increase of AbetaSUB42 and resultant accumulation. In pericytes, ASPD binding to NKAalpha3 releases an unknown effector molecule that activates delta-secretase in intact neurons, further augmenting Abeta42 and producing pathogenic tau1-368 fragment. Thus, Abeta and tau pathologies are directly linked. Stopping the signaling cascades near the origin by inhibiting ASPD-NKAalpha3 interaction (8) may provide a new therapeutic approach.

Repeated Sub-anaesthetic dose of Ketamine Elevates Superoxide Dismutase in Pharmacological Model of Schizophrenia-Like Phenotypes in Mice
Objective: Thestudy evaluatedbehavioural phenotypes and the level of Superioxde Dismutase activity in repeated sub-anaesthetic dose of ketamine administered to model schizophreniain animal study. Method:Animals were divided into groups (n=6), control groupreceived distilled water (10mL/kg)as vehicle (VEH), Ketamine treated group (KET) received sub-anaesthetic dose of ketamine (20mg/kg) for 14 days consecutively.Animals in risperidone treatedgroup(KET+RISP) were pre-treated with sub-anaesthetic dose of ketamine(20mg/kg)alonefor 7 consecutive days, and from day 8-14, risperidone (0.5mg/kg) was administered 1 hour post-ketamine treatment. All treatment were administered intraperitoneally(i.p).Twenty-four (24) hours after the last treatment, Behavioural phenotypes(locomotor activity and cognition)were assessed in locomotor activity cage and elevated maze plus. Thereafter level of superioxde dismutase(SOD)activity was evaluated in homogenized brain tissue of each mouse using spectrophotometric analysis. Result: KET groupshowed significant (p<0.05) increase in movement counts and number of rearing eventsin locomotor activity test , also prolonged latency to enter the open armsin cognitive assessment compared to animals that received distilled water (10mg/kg), and risperidone (0.5mg/kg)treatment. The level of Superioxide Dismutase (SOD) activity was significantly elevated in KET group compare with vehicle control and risperidone treatedanimals. Conclusion: Repeated dose of ketamine may pose differential effect onendogenous antioxidant system which may elevate superioxide dismutase activity in ketamine induced schizophrenic rodent as positive control mechanism.

Age Dependent Integration of Cortical Progenitors Transplanted at the Adult CSF-Brain Interface
There has been renewed interest in neural transplantation of cells and tissues for brain repair. Recent studies have demonstrated the ability of transplanted neural precursor cells and in vitro grown organoids to mature and locally integrate into host brain neural circuitry. Much effort has focused on how the transplant behaves and functions after the procedure, but the extent to which the host brain can properly innervate the transplant, particularly in the context of aging, is largely unexplored. Here we report that transplantation of rat embryonic cortical precursor cells into the cerebrospinal fluid-subventricular zone (CSF-SVZ) of adult rat brains generates a brain-like tissue (BLT) at an ectopic site. This model allows for the measurement of long-range connectivity and cellular interactions between the transplant and the host brain as a function of host age. The transplanted precursor cells initially proliferate, then differentiate, and develop into mature BLTs, which receive supportive cellular components from the host including blood vessels, microglia, astrocytes, and oligodendrocytes. There was integration of the BLT into the host brain which occurred at all ages studied, suggesting that host age does not affect the maturation and integration of the transplant-derived BLT. Long-range axonal projections from the BLT into the host brain were robust throughout the different aged recipients. However, long-distance innervation originating from the host brain into the BLT significantly declined with age. This work demonstrates the feasibility and utility of integrating new neural tissue structures at ectopic sites into adult brain circuits to study host-transplant interactions.

Synaptic connectome of a neurosecretory network in the Drosophila brain
Hormones mediate inter-organ signaling which is crucial in orchestrating diverse behaviors and physiological processes including sleep and activity, feeding, growth, metabolism and reproduction. The pars intercerebralis and pars lateralis in insects represent major hubs which contain neurosecretory cells (NSC) that produce various hormones. To obtain insight into how hormonal signaling is regulated, we have characterized the synaptic connectome of NSC in the adult Drosophila brain. Identification of neurons providing inputs to multiple NSC subtypes implicates diuretic hormone 44-expressing NSC as a major coordinator of physiology and behavior. Surprisingly, despite most NSC having dendrites in the subesophageal zone (primary taste processing center), gustatory inputs to NSC are largely indirect. We also deciphered pathways via which diverse olfactory inputs are relayed to NSC. Further, our analyses revealed substantial inputs from descending neurons to NSC, suggesting that descending neurons regulate both endocrine and motor output to synchronize physiological changes with appropriate behaviors. In contrast to NSC inputs, synaptic output from NSC is sparse and mostly mediated by corazonin NSC. Therefore, we additionally determine putative paracrine interconnectivity between NSC subtypes and hormonal pathways from NSC to peripheral tissues by analyzing single-cell transcriptomic datasets. Our comprehensive characterization of the Drosophila neurosecretory network connectome provides a platform to understand complex hormonal networks and how they orchestrate animal behaviors and physiology.

Columnar cholinergic neurotransmission onto T5 cells of Drosophila
A large variety of acetylcholine receptors (AChRs), nicotinic and muscarinic, are expressed in the brain of Drosophila melanogaster. Nonetheless, how AChRs affect brain function remains poorly understood. T5 cells are the primary motion-sensing neurons in the OFF-motion pathway and receive visual input from four different columnar cholinergic neurons, Tm1, Tm2, Tm4 and Tm9. We reasoned that different postsynaptic AChRs might also contribute to T5 function. We demonstrate that the nicotinic nAChRalpha1, nAChRalpha4, nAChRalpha5 and nAChRalpha7 subunits localize on T5 dendrites. By targeting synaptic markers specifically to each cholinergic input neuron, we uncover a prevalence of the nAChRalpha5 in Tm1, Tm2 and Tm4 synapses and of nAChRalpha7 in Tm9 synapses on T5 dendrites. Knock-down of nAChRalpha4, nAChRalpha5, nAChRalpha7, and mAChR-B individually alters the optomotor response and reduces T5 directional selectivity. Our findings expose the complex molecular mechanisms underlying motion detection in T5 cells and offer a system to study AChRs in visual information processing.