bioRxiv Subject Collection: Neuroscience's Journal

Whole-brain, all-optical interrogation of neuronal dynamics underlying gut interoception in zebrafish
Internal signals from the body and external signals from the environment are processed by brain-wide circuits to guide behavior. However, the complete brain-wide circuit activity underlying interoception - the perception of bodily signals - and its interactions with sensorimotor circuits remain unclear due to technical barriers to accessing whole-brain activity at the cellular level during organ physiology perturbations. We developed an all-optical system for whole-brain neuronal imaging in behaving larval zebrafish during optical uncaging of gut-targeted nutrients and visuo-motor stimulation. Widespread neural activity throughout the brain encoded nutrient delivery, unfolding on multiple timescales across many specific peripheral and central regions. Evoked activity depended on delivery location and occurred with amino acids and D-glucose, but not L-glucose. Many gut-sensitive neurons also responded to swimming and visual stimuli, with brainstem areas primarily integrating gut and motor signals and midbrain regions integrating gut and visual signals. This platform links body-brain communication studies to brain-wide neural computation in awake, behaving vertebrates.

An extracellular complex between CLE-1/collagen XV/XVIII and Punctin/MADD-4 defines cholinergic synapse identity
The precise localization of postsynaptic receptors opposite neurotransmitter release sites is essential for synaptic function. This alignment relies on adhesion molecules, intracellular scaffolds, and a growing class of extracellular scaffolding proteins. However, how these secreted proteins are retained at synapses remains unclear. We addressed this question using C. elegans neuromuscular junctions, where Punctin, a conserved extracellular synaptic organizer, positions postsynaptic receptors. We identified CLE-1, the ortholog of collagens XV/XVIII, as a key stabilizer of Punctin. Punctin and CLE-1B, the main isoform present at neuromuscular junctions, form a complex and rely on each other for synaptic localization. Punctin undergoes cleavage, and in the absence of CLE-1, specific fragments are lost, resulting in the mislocalization of cholinergic receptors to GABAergic synapses. Additionally, CLE-1 regulates receptor levels independently of Punctin. These findings highlight a crucial extracellular complex that maintains synapse identity.

Increased white matter microglial reaction and perivascular macrophages in the aging microcebe primate
Microglia are the resident macrophages of the central nervous system. They play crucial roles in maintaining brain homeostasis, yet their involvement in aging remains not fully understood. While age-related microglia changes have been strongly characterized in rodents, studies in non-human primates are scarce. HLA-DR is a major histocompatibility class II cell surface receptor which presents antigens to cell of the immune response. It is a major marker of microglia reaction. In this study, we explored microglia in the brain of a non-human primate (mouse lemur (Microcebus murinus)) using HLA-DR immunolabeling. We analyzed microglial morphology and quantified HLA-DR+ cell density and protein expression in middle-aged and old animals. A wide range of microglial morphologies was observed in the white matter, including thin processes microglia, rod-like elongated and polarized shape, hypertrophic, and amoeboid microglia. Aging was associated with a region-specific regulation of microglia as increased HLA-DR+ microglial expression was found in the white matter while very few HLA-DR+ microglia were observed in the parenchyma of cortical gray matter regions. A second finding of the study was the higher number of HLA-DR+ perivascular macrophages in old animals. Although further studies are required to investigate the cause of these age-related changes, this study is critical as it describes for the first time the microglial and perivascular macrophage status in microcebus murinus primate that is widely used as a model for cerebral aging and to investigate age-related neurodegenerative processes.

Pre-amyloid cognitive intervention preserves brain function in aged TgF344-AD rats, maintaining connectivity and enhancing plasticity in a sex-specific manner
Background: Alzheimer's disease is characterized by progressive cognitive decline and neurodegeneration, with cognitive reserve playing a key role in mitigating disease impact. Cognitive stimulation has been suggested as a non-pharmacological approach to enhance cognitive reserve and delay cognitive deterioration, but its underlying mechanism remains to be fully understood. This study investigates the effects of pre-amyloid cognitive intervention on brain connectivity, memory, synaptic plasticity and neuroinflammation in aged TgF344-AD rats, considering sex-specific differences. Methods: Male and female TgF344-AD and wild-type rats were assigned to trained and untrained groups, with cognitive stimulation administered through repetitive delayed nonmatch-to-sample tasks. Longitudinal magnetic resonance imaging acquisitions assessed training-induced changes in whole-brain functional connectomics and in particular entorhinal cortex connectivity. Memory was evaluated using the novel object recognition test. Cellular analysis of neurons (NeuN+, Parvalbumin+) and microglial cells, as well as molecular (PSD95, TrkB, p-RPS6, and VGLUT) analyses were conducted to determine the role of cognitive stimulation in modulating neuronal density, neuroinflammation and neuroplasticity. Results: Male TgF344-AD rats undergoing prolonged cognitive stimulation had preserved global functional connectivity and exhibited improved recognition memory, compared to untrained animals, while TgF344-AD females did not follow this pattern. Entorhinal cortex connectivity was significantly loss in 19-month-old rats compared to wild-type rats and this was completely prevented by training. At a cellular level, cognitive stimulation significantly decreased the number of PV+ neurons in the dentate gyrus of trained rats. Moreover, a greater microglial density around beta amyloid plaques and a less reactive phenotype was clearly observed at 11 in trained rats. These protective effects diminished by 19 months, coinciding with increased neuroinflammation and microglial dysfunction. At a molecular level, cognitive stimulation preserved PSD95 expression in male TgF344-AD and p-RPS6 in both sexes. Conclusions: Pre-amyloid cognitive stimulation enhances synaptic plasticity, sustains brain network integrity, and modulates neuroinflammation, contributing to increased resilience against Alzheimer's disease-related cognitive decline. In general, cognitive stimulation exerted a more protective effect in male TgF344-AD rats showing sex-dependent differences in pathology and cognitive reserve mechanisms. These findings highlight the importance of early cognitive engagement as a potential strategy to delay disease onset and underscore sex-specific differences in cognitive resilience mechanisms.

Rare mutations implicate CGE interneurons as a vulnerable axis of cognitive deficits across psychiatric disorders
Neuropsychiatric disorders such as autism spectrum disorder (ASD) and schizophrenia (SCZ) share genetic risk factors, including rare high penetrance single nucleotide variants and copy number variants (CNVs), and exhibit both overlapping and distinct clinical phenotypes. Cognitive deficits and intellectual disability-critical predictors of long-term outcomes-are common to both conditions. To investigate shared and disorder-specific neurobiological impact of highly penetrant rare mutations in ASD and SCZ, we analyzed human single-nucleus whole-brain sequencing data to identify strongly affected brain cell types. Our analysis revealed Caudal Ganglionic Eminence (CGE)-derived GABAergic interneurons as a critical nexus for cognitive deficits across these disorders. Notably, genes within 22q11.2 deletions, known to confer a high risk of SCZ, ASD, and cognitive impairment, showed a strong expression bias toward vasoactive intestinal peptide-expressing cells (VIP+) among CGE subtypes. To explore VIP+ GABAergic interneuron perturbations in the 22q11.2 deletion syndrome in vivo, we examined their activity in the Df(16)A+/- mouse model during a spatial navigation task and observed reduced activity along with altered responses to random rewards. At the population level, VIP+ interneurons exhibited impaired spatial encoding and diminished subtype-specific activity suggesting deficient disinhibition in CA1 microcircuits in the hippocampus, a region essential for learning and memory. Overall, these results demonstrate the crucial role of CGE-derived interneurons in mediating cognitive processes that are disrupted across a range of psychiatric and neurodevelopmental disorders.

Discovery, Interruption, and Updating of Auditory Regularities in Memory: Evidence from Low-Frequency Brain Dynamics in Human MEG
During passive listening, the brain maintains a hierarchy of predictive models to monitor the statistics of its surroundings. The automatic discovery of regular patterns has been associated with a gradual increase in sustained tonic M/EEG activity, sourced in auditory, hippocampal, and frontal areas - reflecting evidence accumulation and establishment of a regularity model. Conversely, when a regular pattern is interrupted, the sustained activity drops - indicating disengagement from the model. However, how such models are established in and retrieved from memory as well as the conditions under which they are activated and interrupted remain underexplored. In this MEG experiment (N=26; both sexes), we examined how neural responses related to model establishment and interruption are influenced by (1) the rate of stimulus presentation (tone presentation rate 20 Hz vs. 40 Hz), and (2) the novelty of the experienced acoustic structure (novel vs resumed REG pattern). The results show that (1) the dynamics of model interruption and establishment are independent of stimulus presentation rate, and that (2) model establishment occurred much faster when an experienced vs novel pattern was presented after pattern interruption, suggesting re-activation of the stored original model. These responses broadly mirrored those of an ideal observer model of prediction. (3) Finally, sustained response rises in response to pattern establishment and interruption were localized in auditory, hippocampal, and frontal sources, supporting top-down model information flow. These results unveil the temporal dynamics and neural network underlying the brain's construction and selection of predictive models to monitor changes in sensory statistics.

Rapid changes in cholinergic signaling, myelination and thyroid signaling pathway gene expression in amygdala subnuclei in response to social status maintenance and reorganization
Male CD-1 mice form linear social hierarchies and can rapidly reform them following social reorganization. Through Tag-based sequencing in the medial amygdala (MeA), we identified several genes regulating cholinergic signaling, myelination, and thyroid signaling that rapidly shift expression 70 minutes after animals change social status. Here, we further characterize the expression patterns of individual genes within these pathways in both stable and reorganized hierarchies. We find that genes related to cholinergic signaling show higher expression in the MeA of dominant males in stable hierarchies as well as when reestablishing dominance in reorganized hierarchies. Dominant males also show higher levels of myelination related genes than socially descending males when reestablishing their social status during social reorganization but less so in stable groups. Conversely, thyroid signaling genes show higher expression in the MeA in subordinate males and previously dominant males who are socially descending. Using RNAscope, we were able to demonstrate broadly similar patterns of gene expression immediately following social reorganization across the MeA, basolateral, and central amygdala for 7 genes of interest (chat, slc5a7, ache, mbp, mog, crym, mybpc1). High levels of co-expression of cholinergic signaling and myelination gene expression in dominant males suggest that these processes work together to promote resilience to the social challenge and promote dominance. In summary, we demonstrate that rapid changes in amygdala gene expression in each pathway are associated with the formation and maintenance of dominance and subordinate social status in stable and reorganized environments.

Oligodendrocyte precursor cells establish regulatory contacts with the soma of active neurons
Oligodendrocyte precursor cells (OPCs) can generate myelinating oligodendrocytes life-long, a process that is dependent on neuronal activity. However, it is possible that OPCs have additional functions, influencing neuronal functions directly. We have used a mouse genetic model of juvenile seizures and chemical induction of neuronal activity to examine the morphological and molecular changes in OPCs around activated principal neurons. We found an increase in process extension of OPCs specifically toward the soma of activated neurons. Moreover, we found that the close proximity of OPC processes around neurons expressing the immediate early gene c-Fos decreased the calcium transients in these neurons, indicating a regulative function of OPCs. Analyses of transcriptome and chromatin accessibility revealed significant changes in genes involved in transforming growth factor beta (TGF beta) signaling. Extracellular matrix genes, particularly those encoding type VI collagen, an established binding partner for the OPC surface protein NG2, was increased around active neurons. Our findings indicate that OPCs are an integral part of the neural network and may help to decrease the activity of neurons that have previously been over-excited, in order to protect these neurons.

Midbrain Glutamatergic Neurons Modulate the Acoustic Startle Reflex and Prepulse Inhibition in Mice
Prepulse inhibition (PPI) of the auditory startle reflex task is a widely recognized operational measure of sensorimotor gating. PPI deficits are a hallmark feature of schizophrenia, often associated with attentional and cognitive impairments. Despite its extensive use in preclinical research for screening antipsychotic drugs, the precise cellular and circuit mechanisms underlying PPI remain unclear, even under physiological conditions. Recent evidence suggests that non-cholinergic inputs from the pedunculopontine tegmental nucleus (PPTg) to the caudal pontine reticular nucleus (PnC) mediate PPI. In this study, we investigated the contribution of PPTg glutamatergic neurons to acoustic startle and PPI. Tract-tracing, immuno-histochemical analyses, and in vitro whole-cell recordings in wild-type mice confirmed that PPTg glutamatergic neurons innervate the PnC. Optogenetic inhibition of PPTg-PnC glutamatergic synapses in vivo resulted in increased PPI across various interstimulus intervals. Notably, while optogenetic activation of this pathway had no additional effect on startle and PPI, activation of this connection alone before startle stimulation reduced startle at short interstimulus intervals and increased startle at longer intervals. Furthermore, although PPTg glutamatergic inputs target PnC glycinergic neurons, our in vitro whole-cell recordings combined with optogenetic stimulation at PPTg-PnC synapses revealed that PPTg glutamatergic inputs activate PnC glutamatergic giant neurons. Our findings identify a feed-forward excitatory mechanism within the brainstem startle circuit, whereby PPTg glutamatergic inputs modulate PnC neuronal activity. These results provide new insights into the clinically relevant theoretical construct of PPI, which is disrupted in various neuropsychiatric and neurological disorders.

Bi-modal microwave neuromodulation via thermal and nonthermal mechanisms
Electrical neuromodulation, the current clinical standard, is invasive, expensive, and prone to malfunction. Electromagnetic waves can perform noninvasive neuromodulation, but existing methods are limited by the tradeoff between penetration depth and spatial precision. Microwaves in the 0.9 - 3 GHz range are widely used for telecommunications and can penetrate to the deep brain. Microwaves have been shown to nonthermally modulate neural activity, but the acute bioeffects remain unclear and under-studied. Here, we employ a microwave rod antenna (MRA) to demonstrate bi-modal neuromodulation via thermal and non-thermal mechanisms. The MRA enabled electrophysiological recordings of neurons exposed to microwaves, which elucidated the differential effects of pulsed and continuous microwaves on neurons. These findings build the foundation for developing microwave-based wireless neuromodulation devices for drug-free treatment of seizures and chronic pain.

Dynamic Ontogeny of Auditory Lateralization in the Zebra Finch
Functional lateralization is a ubiquitous trait in the animal kingdom and represents a general and conserved mechanism of the central nervous system. Lateralized processes observed in adult sensory cortices emerge as a function of development and experience, e.g. speech-processing in humans and processing of conspecific vocalizations in songbirds. Adult Zebra finches (ZFs), a species of songbird, exhibit right-lateralized activity in the higher auditory region caudomedial nidopallium (NCM) which depends on normal rearing conditions; auditory deprivation leads to atypical bilateral responses to conspecific song. Here, we investigate the ontogenetic timeline of auditory lateralization and the lasting effects of auditory rearing-environment in the developing ZF (40-120 days post-hatch, phd). ZFs were raised in one of three acoustic environments: 1) a tutor-playback driven paradigm, 2) chronic exposure to a ZF aviary recording (no tutor), and 3) chronic exposure to a canary aviary recording (no tutor). By longitudinally tracking lateralized auditory evoked potentials at the level of the dura, we show that adult right-lateralized activity 1) emerges from an initial left-biased profile; 2) this left-to-right emergence occurs ~60-80phd and does not require the presence of a tutor; and 3) also emerges in ZFs raised in a canary auditory environment. Furthermore, lateralization and song development were positively correlated, although these measurements are not necessarily causally related. Awake, bilateral NCM electrophysiology in the same birds when adult, confirmed they were right-lateralized and that lateralized activity for specific test stimuli depended on rearing experience. Lastly, a decoding assay showed that canary-based rearing led to increased decoding accuracy of canary test stimuli, suggesting that neurons exhibit enhanced encodability for those sounds heard earlier. Together, the results document the ontogenetic timeline of auditory lateralization in the songbird and show that auditory experience in development, including passive exposure, shapes how auditory regions in the brain process stimuli in adulthood. Taken together with earlier results showing 1) the absence of lateral differences in ZFs reared in auditory deprivation and 2) that lateralization reverses dynamically in concert with improved discrimination in adult ZFs exposed to novel auditory environments, our current timeline suggests that the dynamic emergence of lateralization reflects the brains plastic response to novel auditory experiences.

A language network in the individualized functional connectomes of over 1,000 human brains doing arbitrary tasks
A century and a half of neuroscience has yielded many divergent theories of the neurobiology of language. Two factors that likely contribute to this situation include (a) conceptual disagreement about language and its component processes, and (b) intrinsic inter-individual variability in the topography of language areas. Recent functional magnetic imaging (fMRI) studies of small numbers of intensively scanned individuals have argued that a language-selective brain network emerges from correlations (individualized functional connectomics, iFC) in task-free (e.g., rest) or task-regressed activation timecourses. Here we test this hypothesis at scale and evaluate its practical utility for task-agnostic language localization: we apply iFC separately to each of 1,971 (fMRI) scanning sessions (1,199 unique brains), each consisting of diverse tasks. We find that iFC indeed reveals a left-lateralized frontotemporal network that is more stable within individuals than between them, robust to the granularity of the parcellation, and selective for language. These results support the hypothesis that this network is a key structure in the functional organization of the adult brain and show that it can be recovered retrospectively from arbitrary imaging data, with implications for neuroscience, neurosurgery, and neural engineering.

On a roll: Recent familiarity primes the brain to retrieve other memories via dopaminergic nuclei mechanisms
Memory retrieval is notoriously variable. Various neurocognitive states have been theorized to affect retrieval success from moment to moment, but the presence and catalysts of these states in the human brain remain largely understudied. Building on previous work, we studied how recent memory experiences (i.e. novelty vs. familiarity) and their corresponding neural activity prepare the brain to reactivate memories. Our data uncover a pronounced neural bias, whereby regions representing episodic information fail to reactivate memories following recent experiences of novelty. Drawing on computational models, we hypothesized that novelty-evoked activity in cholinergic nuclei underlie this bias. The data revealed an unexpected alternative mediator of memory reactivation in response to recent novelty, namely recent dopaminergic nuclei activity. By identifying dopamine as a potential mediator of retrieval variability, our results challenge existing models and open new avenues for investigating how neuromodulatory states dynamically shape memory accessibility.

Prior cocaine use disrupts identification of hidden states by single units and neural ensembles in orbitofrontal cortex
The orbitofrontal cortex (OFC) is critical to identifying task structure and to generalizing appropriately across task states with similar underlying or hidden causes. This capability is at the heart of OFCs proposed role in a network responsible for cognitive mapping, and its loss can explain many deficits associated with OFC damage or inactivation. Substance use disorder is defined by behaviors that share much in common with these deficits, such as an inability to modify learned behaviors in the face of new or even anecdotal information about undesired consequences. One explanation for this similarity would be if addictive drugs impacted the ability of OFC to recognize underlying similarities, hidden states, that allow information learned in one setting to be used in another. To explore this possibility, we trained rats to self-administer cocaine and then recorded single unit activity in lateral OFC as these rats performed in an odor sequence task consisting of unique and shared positions. In well-trained controls, we observed chance decoding of sequence at shared positions and near chance decoding even at unique positions, reflecting the irrelevance of distinguishing these positions in the task. By contrast, in cocaine-experienced rats, decoding remained significantly elevated, particularly at the positions that had superficial sensory differences that were collapsed in controls across learning. A tensor component analysis showed that this effect of reduced generalization after cocaine use also extended across positions in the sequences. These results show that prior cocaine use disrupts the normal identification of hidden states by OFC.

Modeling Masticatory Myalgia to Headache-like Referred Pain Triggers Local Gene Plasticity at Referred Pain Sites
Patients with myofascial pain in the head and neck area report widespread and referred pain, including headache. Existing preclinical models fail to replicate this clinical phenotype; therefore, we aimed to develop animal models mimicking referred pain phenomenon and investigate whether referred pain leads to gene plasticity at the referred sites. We modeled masticatory myalgia by stimulation of either the masseter (MM) or temporal muscle (TM) in mice. MM and TM were stimulated with a single high-dose injection of Collagenase-type II (Col), repetitive low-dose Col injections, repetitive gentle MM stimulation, or single or repetitive forceful mouth opening. Referred pain was assessed by measuring mechanical hypersensitivity in the periorbital area (representing headache-like behavior) and another masticatory muscle. Stimulation of the MM, whether through single or repetitive Col injections or mouth opening, produced inconsistent, short-lasting (1-2 days) headache-like behavior in both males and females. In contrast, stimulation of the TM, using different paradigms, triggered mechanical hypersensitivity in both the MM and the periorbital area. Referred headache-like behavior lasted longer in females compared to males, while referred myalgia in the MM was pronouncer in males. The referred pain in the MM and periorbital areas triggered by TM stimulation was associated with significant gene plasticity in the MM and dura mater. Transcriptional changes in the MM following Col injection into the TM resembled those observed after direct MM injections. Presented data imply that referred pain modeled by TM stimulation could be accounted by nociceptive signaling from multiple local sites involved in this referred pain network.

Individual Variation in Intrinsic Neuronal Properties of Nucleus Accumbens Core and Shell Medium Spiny Neurons in Animals Prone to Sign- or Goal-Track
The "sign-tracking" and "goal-tracking" model of individual variation in associative learning permits the identification of rats with different cue-reactivity and predisposition to addiction-like behaviors. Certainly, compared to "goal-trackers" (GTs), "sign-trackers" (STs) show more susceptibility traits such as increased cue-induced 'relapse' of drugs of abuse. Different cue- and reward-evoked patterns of activity in the nucleus accumbens (NAc) have been a hallmark of the ST/GT phenotype. However, it is unknown whether differences in the intrinsic neuronal properties of NAc medium spiny neurons (MSNs) in the core and shell subregions are also a physiological correlate of these phenotypes. We performed whole-cell slice electrophysiology in outbred male rats and found that STs exhibited the lowest excitability in the NAc core, with lower number of action potentials and firing frequency as well as a blunted voltage/current relationship curve in response to hyperpolarized potentials in both the NAc core and shell. Although firing properties of shell MSNs did not differ between STs and GTs, intermediate responders that engage in both behaviors showed greater excitability compared to both STs and GTs. These findings suggest that intrinsic excitability in the NAc may contribute to individual differences in the attribution of incentive salience.

Standardization of postmortem human brainstem along the rostrocaudal axis to accommodate for heterogeneity in samples
Human postmortem brain tissues provide an indispensable resource that is crucial for the understanding of neurological conditions, whether related to pathology subtype, burden, distribution or cell-type specificity. Pathology staging protocols provide guidelines for standardized sampling of brain tissues, but cover only a subset of regions affected by pathologies. Thus, to study how various neuropathologies and cell types in highly specialized circuit nodes correlate with functions specifically served by these nodes, additional protocols are necessary. This especially applies to brainstem tissues due to the small dimension of regions of interest and interindividual variability of specimens, whether due to procurement or intrinsic differences. Here we systematically assessed factors contributing to heterogeneity in the length of whole brainstem samples and then presented a standardized approach to reproducibly assign rostrocaudal levels, with standardization relying upon readily identifiable internal landmarks. We validated this approach using postmortem MRI imaging. Standardized brainstem length correlated positively with subject height and negatively with subject age of death. By providing a reference series, reproducible levels can be assigned to individual histological sections or MRI images, i.e. when full brainstem specimens are not available and irrespective of platform, promoting reproducibility.

Astrocyte and neurogenic mechanisms of protective gene therapy prevent brain disease and death due to non-ketotic hyperglycinemia
Genetic defects in glycine decarboxylase (GLDC) cause non-ketotic hyperglycinemia (NKH), a rare and frequently fatal neurometabolic disease, which lacks FDA-approved therapies. We characterized CRISPR Cas9-edited humanized mice expressing a prevalent clinical mutation after administration with a single intraperitoneal dose of a novel recombinant of adeno-associated viral vector 9 expressing GLDC (rAAV9-GLDC). Long term biological activity of rAAV9-GLDC was first validated by assessment of its systemic efficacy over five and ten months in mice. Access of rAAV9 to the brain was confirmed by tracking green fluorescent protein (GFP) after a single intraperitoneal dose of rAAV9-GFP. Over five months, control "mock" treated GFP-mice showed reduction in astrocytes but not microglia, oligodendrocytes or neurons in the brain. 37% of these animals suffered long term neurological disease and/or death. rAAV9-GLDC boosted novel astrocyte proliferation linked to neurogenesis, in absence of inflammation to confer 100% protection against sickness and fatality due to NKH.