bioRxiv Subject Collection: Neuroscience's Journal

Potentiation of the M1 muscarinic acetylcholine receptor normalizes neuronal activation patterns and improves apnea severity in Mecp2+/- mice
Rett syndrome (RTT) is a neurodevelopmental disorder that is caused by loss-of-function mutations in the methyl-CpG binding protein 2 (MeCP2) gene. RTT patients experience a myriad of debilitating symptoms, which include respiratory phenotypes that are often associated with lethality. Our previous work established that expression of the M1 muscarinic acetylcholine receptor (mAchR) is decreased in RTT autopsy samples, and that potentiation of the M1 receptor improves apneas in a mouse model of RTT; however, the population of neurons driving this rescue is unclear. Loss of Mecp2 correlates with excessive neuronal activity in cardiorespiratory nuclei. Since M1 is found on cholinergic interneurons, we hypothesized that M1-potentiating compounds decrease apnea frequency by tempering brainstem hyperactivity. To test this, Mecp2+/- and Mecp2+/+ mice were screened for apneas before and after administration of the M1 positive allosteric modulator (PAM) VU0453595 (VU595). Brains from the same mice were then imaged for c-Fos, ChAT, and Syto16 using whole-brain light-sheet microscopy to establish genotype and drug-dependent activation patterns that could be correlated with VU595's efficacy on apneas. The vehicle-treated Mecp2+/- brain exhibited broad hyperactivity when coupled with the phenotypic prescreen, which was significantly decreased by administration of VU595, particularly in regions known to modulate the activity of respiratory nuclei (i.e. hippocampus and striatum). Further, the extent of apnea rescue in each mouse showed a significant positive correlation with c-Fos expression in non-cholinergic neurons in the striatum, thalamus, dentate gyrus, and within the cholinergic neurons of the brainstem. These results indicate that Mecp2+/- mice are prone to hyperactivity in brain regions that regulate respiration, which can be normalized through M1 potentiation.

Opposing actions of co-released GABA and neurotensin on the activity of preoptic neurons and on body temperature
Neurotensin (Nts) is a neuropeptide acting as a neuromodulator in the brain. Pharmacological studies have identified Nts as a potent hypothermic agent. The medial preoptic area, a region that plays an important role in the control of thermoregulation, contains a high density of neurotensinergic neurons and Nts receptors. The conditions in which neurotensinergic neurons play a role in thermoregulation are not known. In this study optogenetic stimulation of preoptic Nts neurons induced a small hyperthermia. In vitro, optogenetic stimulation of preoptic Nts neurons resulted in synaptic release of GABA and net inhibition of the preoptic pituitary adenylate cyclase-activating polypeptide (PACAP) neurons firing activity. GABA-A receptor antagonist or genetic deletion of VGAT in Nts neurons unmasked also an excitatory effect that was blocked by a Nts receptor 1 antagonist. Stimulation of preoptic Nts neurons lacking VGAT resulted in excitation of PACAP neurons and hypothermia. Mice lacking VGAT expression in Nts neurons presented changes in the fever response and in the responses to heat or cold exposure as well as an altered circadian rhythm of body temperature. Chemogenetic activation of all Nts neurons in the brain induced a 4-5 {degrees}C hypothermia, which could be blocked by Nts receptor antagonists in the preoptic area. Chemogenetic activation of preoptic neurotensinergic projections resulted in robust excitation of preoptic PACAP neurons. Taken together our data demonstrate that endogenously released Nts can induce potent hypothermia and that excitation of preoptic PACAP neurons is the cellular mechanism that triggers this response.

An optogenetic assay for the dauer decision in Caenorhabditis elegans
The dauer decision in Caenorhabditis elegans is a critical developmental decision that ensures survival under harsh environmental conditions. Factors such as temperature, food availability, and pheromone strongly influence the decision to enter and exit dauer. Traditional assays that assess the dauer decision are often confounded by the influence of pheromones from the population, which are often dynamic and highly variable. To mitigate this issue, we developed a simple, single-housing assay for dauer quantification that is compatible with optogenetics. We show that insulin-like peptides (ILPs) from ASJ and other neurons strongly influence the decision to exit dauer, and that ASJ activity can be manipulated with optogenetics to influence the dauer decision in a temporally precise manner.

Cold induces brain region-selective neuronal activity-dependent lipid metabolism
Previous studies have been focused on lipid metabolism in peripheral tissues such as adipose tissues, while little or nothing is known about that in the brain. It is well recognized that cold acclimations enhance adipocyte functions, including white adipose tissue (WAT) lipid lipolysis and beiging, and brown adipose tissue (BAT) thermogenesis in mammals. However, it remains unclear whether and how the genes responsible for lipid metabolism in the brain are also under the control of cold acclimations. Here, we show that cold exposure predominantly increases the expressions of the genes encoding lipid lipolysis in the paraventricular nucleus of the hypothalamus (PVH). Mechanistically, we find that inactivation of neurons in the PVH blunts the cold-induced lipid peroxidation and lipolysis. Together, these findings indicate that lipid metabolism in the PVH is cold sensitive, potentially participating in cold regulations of energy metabolism.

Potent and reversible open-channel blocker of NMDA receptor derived from dizocilpine with enhanced membrane-to-channel inhibition
N-methyl-D-aspartate receptors (NMDARs) play a significant role in developing several central nervous system (CNS) disorders. Currently, memantine, used for treating Alzheimer's disease, and ketamine, known for its anesthetic and antidepressant properties, are two clinically used NMDAR open-channel blockers. However, despite extensive research into NMDAR modulators, many have shown either harmful side effects or inadequate effectiveness. For instance, dizocilpine (MK-801) is recognized for its powerful psychomimetic effects due to its high-affinity and nearly irreversible inhibition of the GluN1/GluN2 NMDAR subtypes. Unlike ketamine, memantine and MK-801 also act through a unique, low-affinity "membrane-to-channel inhibition" (MCI). We aimed to develop an open-channel blocker based on MK-801 with distinct inhibitory characteristics from memantine and MK-801. Our novel compound, K2060, demonstrated effective voltage-dependent inhibition in the micromolar range at key NMDAR subtypes, GluN1/GluN2A and GluN1/GluN2B, even in the presence of Mg2+. K2060 showed reversible inhibitory dynamics and a partially trapping open-channel blocking mechanism with a significantly stronger MCI than memantine. Using hippocampal slices, 30 uM K2060 inhibited excitatory postsynaptic currents in CA1 hippocampal neurons by ~51%, outperforming 30 uM memantine (~21% inhibition). K2060 exhibited No Observed Adverse Effect Level (NOAEL) of 15 mg/kg upon intraperitoneal administration in mice. Administering K2060 at a 10 mg/kg dosage resulted in brain concentrations of approximately 2 uM, with peak concentrations (Tmax) achieved within 15 minutes. Finally, applying K2060 with trimedoxime and atropine in mice exposed to tabun improved treatment outcomes. These results underscore K2060's potential as a therapeutic agent for CNS disorders linked to NMDAR dysfunction.

Recurrent Interneuron Connectivity does not Support Synchrony in a Biophysical Dentate Gyrus Model
Synchronous activity of neuronal networks is found in many brain areas and correlates with cognition and behavior. Gamma synchrony is particularly strong in the dentate gyrus, which is thought to process contextual information in the hippocampus. Several network mechanisms for synchrony generation have been proposed and studied computationally. One such mechanism relies solely on recurrent inhibitory interneuron connectivity, but it requires a large enough number of synapses. Here, we incorporate connectivity data of the dentate gyrus into a biophysical computational model to test its ability to generate synchronous activity. We find that recurrent interneuron connectivity is insufficient to induce a synchronous network state. This applies to an interneuron ring network and the broader dentate gyrus circuitry. In the asynchronous state, recurrent interneuron connectivity can have small synchronizing effects but can also desynchronize the network for specific types of synaptic input. Our results show that synchronizing mechanisms relying solely on interneurons are unlikely to be biologically plausible in the dentate gyrus.

Single molecule array measures of LRRK2 kinase activity in serum link Parkinson's disease severity to peripheral inflammation
Background: LRRK2-targeting therapeutics that inhibit LRRK2 kinase activity have advanced to clinical trials in idiopathic Parkinson's disease (iPD). LRRK2 phosphorylates Rab10 on endolysosomes in phagocytic cells to promote some types of immunological responses. The identification of factors that regulate LRRK2-mediated Rab10 phosphorylation in iPD, and whether phosphorylated-Rab10 levels change in different disease states, or with disease progression, may provide insights into the role of Rab10 phosphorylation in iPD and help guide therapeutic strategies targeting this pathway. Methods: Capitalizing on past work demonstrating LRRK2 and phosphorylated-Rab10 interact on vesicles that can shed into biofluids, we developed and validated a high-throughput single-molecule array assay to measure extracellular pT73-Rab10. Ratios of pT73-Rab10 to total Rab10 measured in biobanked serum samples were compared between informative groups of transgenic mice, rats, and a deeply phenotyped cohort of iPD cases and controls. Multivariable and weighted correlation network analyses were used to identify genetic, transcriptomic, clinical, and demographic variables that predict the extracellular pT73-Rab10 to total Rab10 ratio. Results: pT73-Rab10 is absent in serum from Lrrk2 knockout mice but elevated by LRRK2 and VPS35 mutations, as well as SNCA expression. Bone-marrow transplantation experiments in mice show that serum pT73-Rab10 levels derive primarily from circulating immune cells. The extracellular ratio of pT73-Rab10 to total Rab10 is dynamic, increasing with inflammation and rapidly decreasing with LRRK2 kinase inhibition. The ratio of pT73-Rab10 to total Rab10 is elevated in iPD patients with greater motor dysfunction, irrespective of disease duration, age, sex, or the usage of PD-related or anti-inflammatory medications. pT73-Rab10 to total Rab10 ratios are associated with neutrophil activation, antigenic responses, and the suppression of platelet activation. Conclusions: The extracellular ratio of pT73-Rab10 to total Rab10 in serum is a novel pharmacodynamic biomarker for LRRK2-linked innate immune activation associated with disease severity in iPD. We propose that those iPD patients with higher serum pT73-Rab10 levels may benefit from LRRK2-targeting therapeutics to mitigate associated deleterious immunological responses.

Using rapid invisible frequency tagging (RIFT) to probe the attentional distribution between speech planning and comprehension
Interlocutors often use the semantics of comprehended speech to inform the semantics of planned speech. Do representations of the comprehension and planning stimuli interact on a neural level? We used rapid invisible frequency tagging (RIFT) and EEG to probe the attentional distribution between spoken distractor words and target pictures in the picture-word interference (PWI) paradigm. We presented participants with auditory distractor nouns (auditory (f1); tagged at 54Hz) together with categorically related or unrelated pictures (visual (f2); tagged at 68Hz), which had to be named after a delay. RIFT elicits steady-state evoked potentials, which reflect attentional allocation to the tagged stimuli. When representations of the tagged stimuli interact, integrative effects have been observed at the intermodulation frequency resulting from an interaction of the base frequencies (f2-f1 or f2+f1; Drijvers et al., 2021). Our results showed clear power increases at 54Hz and 68Hz during the tagging window, but no differences between related or unrelated conditions. More interestingly, we observed a larger power difference in the unrelated compared to the related condition at the intermodulation frequency (68Hz-54Hz: 14Hz), indicating stronger interaction between the auditory and visual representations when they were unrelated. Our results go beyond standard PWI results (e.g., Burki et al., 2020) by showing that participants do not have more difficulty visually attending to the related pictures or inhibiting the related auditory distractors. Instead, processing difficulties arise when the representations of the stimuli interact, meaning that participants might be trying to prevent integration between the auditory and visual representations in the related condition.

Anatomical and functional studies of isolated vestibular neuroepithelia from Meniere's Disease patients
Surgical removal of vestibular end organs is a final treatment option for people with intractable Meniere's Disease. We describe the use of surgically excised vestibular neuroepithelium from patients with Meniere's Disease for 1) anatomical investigation of hair cell and nerve fibres markers using immunohistochemistry and 2) functional studies using electrophysiological recordings of voltage-activated currents. Our data shows considerable reduction in and disorganization of vestibular hair cells in the cristae ampullares while nerve fibres are in contact with remaining sensory receptors but appear thin in regions where hair cells are absent. Electrophysiological recordings of voltage-activated potassium currents from surviving hair cells demonstrate normal activity in both type I and type II vestibular hair cells. In addition, current-voltage plots from type I vestibular hair cells are consistent with the presence of a surrounding calyx afferent terminal. These data indicate surviving hair cells in Meniere's Disease patients remain functional and capable of transmitting sensory information to the central nervous system. Determining functionality of vestibular receptors and nerves is critical for vestibular implant research to restore balance in people with Meniere's Disease.

Dysconnectivity of the cerebellum and somatomotor network correlates with the severity of alogia in chronic schizophrenia
Recent fMRI resting-state findings show aberrant functional connectivity within somatomotor network (SMN) in schizophrenia. Moreover, functional connectivity aberrations of the motor system are often reported to be related to the severity of psychotic symptoms. Thus, it is important to validate those findings and confirm their relationship with psychopathology. Therefore, we decided to take an entirely data-driven approach in our fMRI resting-state study of 30 chronic schizophrenia outpatients and 30 matched control subjects. We used independent component analysis (ICA), dual regression, and seed-based connectivity analysis. We found reduced functional connectivity within SMN in schizophrenia patients compared to controls and SMN hypoconnectivity with the cerebellum in schizophrenia patients. The latter is strongly correlated with the intensity of alogia, i.e. poverty of speech and reduction in spontaneous speech, demonstrated by patients. Our results are consistent with the recent knowledge about the role of the cerebellum in cognitive functioning and its abnormalities in psychiatric disorders, e.g. schizophrenia. In conclusion, the presented results, for the first time clearly showed the involvement of the cerebellum hypoconnectivity with SMN in the persistence and severity of alogia symptoms in schizophrenia.

SARS-CoV2 infection triggers reactive astrocyte states and inflammatory conditions in long-term Human Cortical Organoids
SARS-CoV2, severe acute respiratory syndrome coronavirus 2, is frequently associated with neurological manifestations. Despite the presence of mild to severe CNS-related symptoms in a cohort of patients, there is no consensus whether the virus can infect directly brain tissue or if the symptoms in patients are a consequence of peripheral infectivity of the virus. Here, we use long-term human stem cell-derived cortical organoids to assess SARS-CoV2 infectivity of brain cells and unravel the cell-type tropism and its downstream pathological effects. Our results show consistent and reproducible low levels of SARS-CoV2 infection of astrocytes, deep projection neurons, upper callosal neurons and inhibitory neurons in 6 months human cortical organoids. Interestingly, astrocytes showed the highest infection rate among all infected cell populations that led to increased presence of reactive states. Further, transcriptomic analysis revealed overall changes in expression of genes related to cell metabolism, astrocyte activation and, inflammation and further, upregulation of cell survival pathways. Thus, local and minor infectivity of SARS-CoV2 in the brain may induce widespread adverse effects and may lead to resilience of dysregulated neurons and astrocytes within an inflammatory environment.

Stress in utero: effects on gliovascular integrity in female infant offspring
From early in life, experiences like prenatal stress profoundly affect long-term health and behavior. Maternal stress increases fetal exposure to glucocorticoids (GC), disrupting neurodevelopment and raising susceptibility to psychiatric disorders. Previous studies on synthetic GCs, such as dexamethasone (DEX), revealed impairments in neurogenesis and dendritic spine development. However, the influence of prenatal stress on the gliovascular interface remains unclear. This interface, involving the relationship between astrocytes and blood vessels, is essential for healthy brain development. Our study demonstrates that prenatal stress alters the expression and localization of astrocytic proteins crucial for maintaining vascular homeostasis, such as aquaporin-4, in female offspring exposed to DEX. While overall vascular density remains unaffected, it triggers morphological changes. Particularly, the hippocampus and prefrontal cortex exhibit heightened vulnerability to these effects. This study reveals prenatal stress as a potent disruptor of gliovascular development, urging deeper inquiry into its implications.

Roles of the 5-HT1A receptor in zebrafish responses to potential threat and in sociality
Anxiety is a normal emotion representing a reaction to potential danger, whereas fear can be defined as a reaction to real, explicit danger. Anxiety-like behavior in animal models has been associated with differences in the serotonergic system. Treatment of zebrafish cohorts with 8-OH-DPAT, a full agonist at the 5-HT1A receptor, decreased anxiety-like behavior in the novel tank test, but increased it in the light-dark preference test, both considered assays for anxiety-like behavior for this species. The same treatment decreased social approach in both the social investigation and social novelty phases of the social preference test. These effects suggest a participation of the 5-HT1A heteroreceptors in zebrafish anxiety and social preference, decreasing both. Thus, the study of this receptor is important for a better understanding of anxiety-like behavior in zebrafish and its relationship with similar phenomena in vertebrates.

Beyond oscillations - A novel feature space for characterizing brain states
Our moment-to-moment conscious experience is paced by transitions between states, each one corresponding to a change in the electromagnetic brain activity. One consolidated analytical choice is to characterize these changes in the frequency domain, such that the transition from one state to the other corresponds to a difference in the strength of oscillatory power, often in pre-defined, theory-driven frequency bands of interest. Today, the huge leap in available computational power allows us to explore new ways to characterize electromagnetic brain activity and its changes. Here we leveraged an innovative set of features on an MEG dataset with 29 human participants, to test how these features described some of those state transitions known to elicit prominent changes in the frequency spectrum, such as eyes-closed vs eyes-open resting-state or the occurrence of visual stimulation. We then compared the informativeness of multiple sets of features by submitting them to a multivariate classifier (SVM). We found that the new features outperformed traditional ones in generalizing states classification across participants. Moreover, some of these new features yielded systematically better decoding accuracy than the power in canonical frequency bands that has been often considered a landmark in defining these state changes. Critically, we replicated these findings, after pre-registration, in an independent EEG dataset (N=210). In conclusion, the present work highlights the importance of a full characterization of the state changes in the electromagnetic brain activity, which takes into account also other dimensions of the signal on top of its description in theory-driven frequency bands of interest.

Interneuron FGF13 regulates seizure susceptibility via a sodium channel-independent mechanism
Developmental and Epileptic Encephalopathies (DEEs), a class of devastating neurological disorders characterized by recurrent seizures and exacerbated by disruptions to excitatory/inhibitory balance in the brain, are commonly caused by mutations in ion channels. Disruption of, or variants in, FGF13 were implicated as causal for a set of DEEs, but the underlying mechanisms were clouded because FGF13 is expressed in both excitatory and inhibitory neurons, FGF13 undergoes extensive alternative splicing producing multiple isoforms with distinct functions, and the overall roles of FGF13 in neurons are incompletely cataloged. To overcome these challenges, we generated a set of novel cell type-specific conditional knockout mice. Interneuron-targeted deletion of Fgf13 led to perinatal mortality associated with extensive seizures and impaired the hippocampal inhibitory/excitatory balance while excitatory neuron-targeted deletion of Fgf13 caused no detectable seizures and no survival deficits. While best studied as a voltage-gated sodium channel (Nav) regulator, we observed no effect of Fgf13 ablation in interneurons on Navs but rather a marked reduction in K+ channel currents. Re-expressing different Fgf13 splice isoforms could partially rescue deficits in interneuron excitability and restore K+ channel current amplitude. These results enhance our understanding of the molecular mechanisms that drive the pathogenesis of Fgf13-related seizures and expand our understanding of FGF13 functions in different neuron subsets.

Hippocampal place cell sequences during a visual discrimination task: recapitulation of paths near the chosen reward site and independence from perirhinal activity
Compressed hippocampal place-cell sequences have been associated with memory storage, retrieval and planning, but it remains unclear how they align with activity in the parahippocampal cortex. In a visuospatial discrimination task, we found a wide repertoire of hippocampal place cell sequences, which recapitulated paths across the task environment. Place cell sequences generated at reward sites predominantly reiterated trajectories near the chosen maze side, whereas trajectories associated with the side chosen in the previous trial were underrepresented. We hypothesized that neurons in the perirhinal cortex, which during the task display broad firing fields correlated with the animal's location, might reactivate in concert with hippocampal sequences. However, we found no evidence of significant perirhinal engagement during virtual trajectories, indicating that these hippocampal memory-related operations can occur independently of the perirhinal cortex.

Elevated body mass index in youth is associated with dysregulated surrogate markers of neural inhibition & excitation, and internetwork functional dysconnectivity
The developing child and adolescent brain is thought to have an increased vulnerability to the negative impact of obesity and excessive consumption of hyperpalatable and energy-rich foods. In this study, we investigated the neurophysiological effects of overweight and obesity in 30 participants spanning childhood and adolescence (8-19 years), using a naturalistic viewing paradigm with Inscapes, in a pseudo-resting-state protocol scan with magnetoencephalography (MEG). Subjects were median split on body mass indices (BMI), categorised into two groups comprising: lower <25 kg/m2 (n=15) and greater than or equal to 25 kg/m2 (n=15). We assessed spontaneous, regional neural function indexed by oscillatory activity, and functional connectivity within and between intrinsic resting brain networks, including the default mode network, dorsal and ventral attention, somatomotor, visual, language, central executive and salience networks. Elevated BMI was associated with significant reductions in activity of the posterior dominant rhythm, and gamma hyperactivity across widespread cortical areas, suggesting intrinsic neuronal hyperexcitability and disinhibition in children and adolescents. Additionally, we observed low-frequency theta hypoconnectivity between resting state networks including the salience, visual, and default mode networks, and overall reduced global efficiency in brain network structure, suggesting reduced effectiveness in neural communication. These findings underscore the neural impact of body composition on the developing brain, suggesting deleterious alterations in excitation and inhibition from surrogate neural markers associated with neurochemistry and brain networks linked with cognitive and behavioural functioning. These alterations may contribute to the persistent behavioural rigidity and difficulties in adopting healthier eating behaviours into adulthood.

A minimal presynaptic protein machinery mediating synchronous and asynchronous exocytosis and short-term plasticity.
Neurotransmitters are released from synaptic vesicles with remarkable precision in response to presynaptic Ca2+ influx but exhibit significant heterogeneity in exocytosis timing and efficacy based on the recent history of activity. This heterogeneity is critical for information transfer in the brain, yet its molecular basis remains poorly understood. Here, we employ a biochemically-defined fusion assay under physiologically-relevant conditions to delineate the minimal protein machinery sufficient to account for different modes of Ca2+-triggered vesicle fusion and short-term facilitation. We find that Synaptotagmin-1, Synaptotagmin-7, and Complexin, synergistically restrain SNARE complex assembly, thus preserving vesicles in a stably docked state at rest. Upon Ca2+ activation, Synaptotagmin-1 induces rapid vesicle fusion, while Synaptotagmin-7 mediates delayed fusion. Competitive binding of Synaptotagmin-1 and Synaptotagmin-7 to the same SNAREs, coupled with differential rates of Ca2+-triggered fusion clamp reversal, govern the kinetics of vesicular fusion. Under conditions mimicking sustained neuronal activity, the Synaptotagmin-7 fusion clamp is destabilized by the elevated basal Ca2+ concentration, thereby enhancing the synchronous component of fusion. These findings provide a direct demonstration that a small set of proteins is sufficient to account for how nerve terminals adapt and regulate the Ca2+-evoked neurotransmitter exocytosis process to support their specialized functions in the nervous system.

Connectome-driven neural inventory of a complete visual system
Vision provides animals with detailed information about their surroundings, conveying diverse features such as color, form, and movement across the visual scene. Computing these parallel spatial features requires a large and diverse network of neurons, such that in animals as distant as flies and humans, visual regions comprise half the brain's volume. These visual brain regions often reveal remarkable structure-function relationships, with neurons organized along spatial maps with shapes that directly relate to their roles in visual processing. To unravel the stunning diversity of a complex visual system, a careful mapping of the neural architecture matched to tools for targeted exploration of that circuitry is essential. Here, we report a new connectome of the right optic lobe from a male Drosophila central nervous system FIB-SEM volume and a comprehensive inventory of the fly's visual neurons. We developed a computational framework to quantify the anatomy of visual neurons, establishing a basis for interpreting how their shapes relate to spatial vision. By integrating this analysis with connectivity information, neurotransmitter identity, and expert curation, we classified the ~53,000 neurons into 727 types, about half of which are systematically described and named for the first time. Finally, we share an extensive collection of split-GAL4 lines matched to our neuron type catalog. Together, this comprehensive set of tools and data unlock new possibilities for systematic investigations of vision in Drosophila, a foundation for a deeper understanding of sensory processing.