bioRxiv Subject Collection: Neuroscience's Journal

Escalation of intravenous fentanyl self-administration and assessment of withdrawal behavior in male and female mice
Background: The rise in overdose deaths from synthetic opioids, especially fentanyl, necessitates the development of preclinical models to study fentanyl use disorder (FUD). While there has been progress with rodent models, additional translationally relevant models are needed to examine excessive fentanyl intake and withdrawal symptoms. Methods: The study performed intravenous self-administration (IVSA) of fentanyl in male and female C57BL/6J mice for 14 days. Mechanical pain sensitivity during withdrawal was assessed using the von Frey test. Anxiety-like behavior was evaluated via the open field test one-week into abstinence and incubation of craving for fentanyl was assessed four weeks into abstinence. Results: Both male and female mice demonstrated a significant escalation in fentanyl intake over the 14 days of self-administration, with significant front-loading observed in the final days of self-administration. Increased mechanical pain sensitivity was present from 36- to 48-hour into withdrawal and increased anxiety-like behavior was found 1 week into abstinence. Four weeks into abstinence, mice showed significantly higher active lever pressing than the final self-administration session prior to abstinence. Conclusions: The study establishes a translationally relevant mouse model of IVSA of fentanyl, effectively encapsulating critical aspects of FUD, including escalation of drug intake, front-loading behavior, withdrawal symptoms, and prolonged craving for drug into abstinence. This model offers a robust basis for further exploration into behavioral and neurobiological mechanisms involved in fentanyl dependence and potential therapeutic strategies.

Generation of hiPSC-derived brain microvascular endothelial cells using a combination of directed differentiation and transcriptional reprogramming strategies
The blood-brain barrier (BBB), formed by specialized brain microvascular endothelial cells (BMECs), regulates brain function in health and disease. In vitro modeling of the human BBB is limited by the lack of robust protocols to generate BMECs from human iPSCs (hiPSCs). Here, we report generation of reprogrammed BMECs (rBMECs) through combining hiPSC differentiation into BBB-primed endothelial cells (bpECs) and reprogramming with two BBB transcription factors, FOXF2 and ZIC3. rBMECs express a subset of the BBB gene repertoire including tight junctions and transporters, exhibit higher paracellular barrier properties, lower caveolar-mediated transcytosis, and equivalent p-glycoprotein activity compared to primary HBMECs, and can be activated by oligomeric A{beta}42. We then generated an hiPSC-derived 3D neurovascular system that incorporates rBMECs, pericytes, and astrocytes using the MIMETAS platform. This novel 3D system closely resembles the in vivo BBB at structural and functional levels and can be used to study pathogenic mechanisms of neurological diseases.

Ketamine can produce oscillatory dynamics by engaging mechanisms dependent on the kinetics of NMDA receptors
Ketamine is an NMDA-receptor antagonist that produces sedation, analgesia and dissociation at low doses and profound unconsciousness with antinociception at high doses. At high and low doses, ketamine can generate gamma oscillations (>25 Hz) in the electroencephalogram (EEG). The gamma oscillations are interrupted by slow-delta oscillations (0.1-4 Hz) at high doses. Ketamine's primary molecular targets and its oscillatory dynamics have been characterized. However, how the actions of ketamine at the subcellular level give rise to the oscillatory dynamics observed at the network level remains unknown. By developing a biophysical model of cortical circuits, we demonstrate how NMDA-receptor antagonism by ketamine can produce the oscillatory dynamics observed in human EEG recordings and non-human primate local field potential recordings. We have discovered how impaired NMDA-receptor kinetics can cause disinhibition in neuronal circuits and how a disinhibited interaction between NMDA-receptor-mediated excitation and GABA-receptor-mediated inhibition can produce gamma oscillations at high and low doses, and slow-delta oscillations at high doses. Our work uncovers general mechanisms for generating oscillatory brain dynamics that differs from ones previously reported, and provides important insights into ketamine's mechanisms of action as an anesthetic and as a therapy for treatment-resistant depression.

Cardiac Cycle Modulates Alpha and Beta Suppression during Motor Imagery.
Introduction The baroreceptor hypothesis posits that baroreceptors, located on the cardiac walls, are most active during systole, translating cardiac contraction information to the brain. Studies within this context have suggested that the systolic phase, characterised by increased noise, may compromise the processing of sensory stimuli. Although the effect of systolic and diastolic cardiac cycle phases on cognition, perception, and action has been widely documented, there remains a gap in applying these interoceptive insights to enhance assistive technologies such as brain-computer interfaces (BCIs). In the context of BCIs, motor imagery (MI), the mental rehearsal of movement, serves as a widely used control paradigm, yet its modulation through the cardiac cycle has not been empirically tested. Bridging this gap, this study examined how the cardiac cycle phases influence MI by assessing their effect on contralateral suppression of alpha (8-13 Hz) and beta (14-30 Hz) activity in primary sensorimotor cortices. Materials & Methods Twenty-nine participants performed left/right thumb abductions based on the direction of an arrow presented on the screen to get familiarised with kinesthetic sensations. They then completed a MI task of the same movements. We recorded both electroencephalography (EEG) and electrocardiography (ECG), focusing our analysis on data epochs aligned with the experimental cue, based on whether it occurred during the systolic or diastolic phase of the cardiac cycle. Time-frequency analysis of source-reconstructed data assessed cue-induced changes in power spectral density (PSD) within the alpha and beta bands in the postcentral and precentral gyrus. Results We found that alpha and beta suppression in the contralateral primary motor and somatosensory cortex was more pronounced when the cue fell during the diastolic phase of the cardiac cycle than during the systolic phase. Validating the main results, an analysis with circular statistics revealed that trials with particularly pronounced contralateral alpha and beta suppression featured cues with latencies clustering during diastole, the quietest time of the cardiac cycle. Accompanying the EEG effects, EMG activity on the side of the movement was enhanced during diastole. Conclusion These findings provide evidence that MI performance can be enhanced by considering the cardiac cycle phases, offering promising implications for BCI-based applications.

Selective engagement of prefrontal VIP neurons in reversal learning
To gain insights into neural mechanisms enabling behavioral adaptations to complex and multidimensional environmental dynamics, we examined roles of VIP neurons in mouse medial prefrontal cortex (mPFC) in probabilistic reversal learning. Behaviorally, manipulating VIP neuronal activity left probabilistic classical conditioning unaffected but severely impaired reversal learning. Physiologically, conditioned cue-associated VIP neuronal responses changed abruptly after encountering an unexpected reward. They also conveyed strong reward prediction error signals during behavioral reversal, but not before or after, unlike pyramidal neurons which consistently conveyed error signals throughout all phases. Furthermore, the signals persistence across trials correlated with reversal learning duration. These results suggest that mPFC VIP neurons play crucial roles in rapid reversal learning, but not in incremental cue-outcome association learning, by monitoring significant deviations from ongoing environmental contingency and imposing error-correction signals during behavioral adjustments. These findings shed light on the intricate cortical circuit dynamics underpinning behavioral flexibility in complex, multifaceted environments.

Prominent involvement of acetylcholine in shaping stable olfactory representation across the Drosophila brain
Despite the vital role of neuromodulation in the neural system, the specific spatiotemporal dynamics of neuromodulators and their interactions with neuronal activities in vivo are still unclear, hampering our understanding of their information representation and functional contributions systemically. To address this problem, we employed two-photon synthetic aperture microscopy (2pSAM) to record long-term neuronal and neuromodulatory olfactory responses across the Drosophila brain at high speed. Our results revealed distinct response properties, global information propagation, functional connectivity, and odor identity representation among neuronal, cholinergic, and serotoninergic dynamics across multiple brain regions. We discovered the compensation between neuronal activity and cholinergic dynamics, both in the odor identity representation across the brain and the functional connectivity network structures of specific brain regions. Moreover, employing low-dimensional manifold and functional connectivity network analyses, we characterized the stable representation of cholinergic dynamics over a long term. Collectively, our unbiased and comprehensive investigation unveiled the prominent involvement of acetylcholine (ACh) in shaping olfactory representation across the brain, underscoring the inadequacy of solely considering neuronal activities when examining information representation of the brain.

Proactive distractor suppression in early visual cortex
Avoiding distraction by salient yet irrelevant stimuli is critical when accomplishing daily tasks. One possible mechanism to accomplish this is by suppressing stimuli that may be distracting such that they no longer compete for attention. While the behavioral benefits of distractor suppression are well-established, its neural underpinnings are not yet fully understood. In an fMRI study, we examined whether and how sensory responses in early visual areas show signs of distractor suppression after incidental learning of spatial statistical regularities. Participants were exposed to an additional singleton task where, unbeknownst to them, one location more frequently contained a salient distractor. We then analyzed whether visual responses in terms of fMRI BOLD were modulated by this distractor predictability. Our findings indicate that implicit spatial priors shape sensory processing even at the earliest stages of cortical visual processing, evident in early visual cortex as a suppression of stimuli at locations which frequently contained distracting information. Notably, while this suppression was spatially (receptive field) specific, it did extend to nearby neutral locations, and occurred regardless of whether the distractor, a nontarget item or the target was presented at this location, suggesting that suppression arises before stimulus identification. Crucially, we observed a similar pattern of spatially specific neural suppression even if search was only anticipated, but no search display was presented. Our results highlight proactive modulations in early visual cortex, where potential distractions are suppressed preemptively, before stimulus onset, based on learned expectations. Combined, our study underscores how the brain leverages implicitly learned prior knowledge to optimize sensory processing and attention allocation.

MODERATE ALCOHOL CONSUMPTION INDUCES LASTING IMPACTS ON PREFRONTAL CORTICAL SIGNALING IN MICE
Both alcohol use disorder (AUD) and Alzheimer's Disease and Related Dementias (ADRD) appear to include disruption in the balance of excitation and inhibition in the cortex, but their potential interactions are unclear. We examined the effect of moderate voluntary binge alcohol consumption on the aged, pre-disease neuronal environment by measuring intrinsic excitability and spontaneous neurotransmission on prefrontal cortical pyramidal (excitatory, glutamatergic) and non-pyramidal (inhibitory, GABAergic) neurons following a prolonged period of abstinence from alcohol in mice. Results highlight that binge alcohol consumption has lasting impacts on the electrophysiological properties of prefrontal cortical neurons. A profound increase in excitatory events onto layer 2/3 non-pyramidal neurons following alcohol consumption was seen, along with altered intrinsic excitability of pyramidal neurons, which could have a range of effects on Alzheimer's Disease progression in humans. These results indicate that moderate voluntary alcohol influences the pre-disease environment in aging and highlight the need for further mechanistic investigation into this risk factor.

Conservation of cortical crowding distance across individuals in human V4
Crowding is the failure to recognize an object due to insufficient spacing, slowing daily tasks such as reading and search. Across 49 observers, we found large variations in psychophysical crowding distance and retinotopic map size. These measures covary, conserving a 1.4-mm cortical crowding distance (threshold object spacing on the cortical surface) in the human V4 map, but not V1-V3, linking the spacing limit of visual recognition to overall V4 size.

Gαolf Regulates Biochemical Signaling in Neurons Associated with Movement Control and Initiation
The heterotrimeric G-protein subunit, Golf, acts to transduce extracellular signals through G-protein coupled receptors (GPCRs) and stimulates adenylyl cyclase mediated production of the second messenger cyclic adenosine monophosphate. Numerous mutations in the GNAL gene, which encodes Golf, have been identified as causative for an adult-onset dystonia. These mutations disrupt GPCR signaling cascades in in vitro assays through several mechanisms, and this disrupted signaling is hypothesized to lead to dystonic motor symptoms in patients. However, the cells and circuits that mutations in GNAL corrupt are not well understood. Published patterns of Golf expression outside the context of the striatum are sparse, conflicting, often lack cell type specificity, and may be confounded by expression of the close GNAL homolog of GNAS. Here, we use RNAScope in-situ hybridization to quantitatively characterize Gnal mRNA expression in brain tissue from wildtype C57BL/6J adult mice. We observed widespread expression of Gnal puncta throughout the brain, suggesting Golf is expressed in more brain structures and neuron types than previously accounted for. We quantify transcripts at a single cell level, and use neuron type specific markers to further classify and understand patterns of GNAL expression. Our data suggests that brain regions classically associated with motor control, initiation, and regulation show the highest expression of GNAL, with Purkinje Cells of the cerebellum showing the highest expression of any neuron type examined. Subsequent Gnal knockout in Purkinje cells led to markedly decreased intracellular cAMP levels and downstream cAMP-dependent enzyme activation. Our work provides a detailed characterization of Gnal expression throughout the brain and the biochemical consequences of loss of Golf signaling in vivo in neurons that highly express Gnal.

Endogenous hydrogen peroxide positively regulates secretion of a gut-derived peptide in neuroendocrine potentiation of the oxidative stress response in C. elegans
The gut-brain axis mediates bidirectional signaling between the intestine and the nervous system and is critical for organism-wide homeostasis. Here we report the identification of a peptidergic endocrine circuit in which bidirectional signaling between neurons and the intestine potentiates the activation of the antioxidant response in C. elegans. We identify a FMRF-amide-like peptide, FLP-2, whose release from the intestine is necessary and sufficient to activate the intestinal oxidative stress response by promoting the release of the antioxidant FLP-1 neuropeptide from neurons. FLP-2 secretion from the intestine is positively regulated by endogenous hydrogen peroxide (H2O2) produced in the mitochondrial matrix by sod-3/superoxide dismutase, and is negatively regulated by prdx-2/peroxiredoxin, which depletes H2O2 in both the mitochondria and cytosol. H2O2 promotes FLP-2 secretion through the DAG and calcium-dependent protein kinase C family member pkc-2 and by the SNAP25 family member aex-4 in the intestine. Together, our data demonstrate a role for intestinal H2O2 in promoting inter-tissue antioxidant signaling through regulated neuropeptide-like protein exocytosis in a gut-brain axis to activate the oxidative stress response.

Ketamine metabolism via hepatic CYP450 isoforms contributes to its sustained antidepressant actions.
(R,S)-ketamine (ketamine) has rapid and sustained antidepressant (AD) efficacy at sub-anesthetic doses in depressed patients. A metabolite of ketamine, including (2R,6R)-hydroxynorketamine (6)-HNKs has been reported to exert antidepressant actions in rodent model of anxiety/depression. To further understand the specific role of ketamine metabolism in the AD actions of the drug, we evaluated the effects of inhibiting hepatic cytochrome P450 enzymes on AD responses. We assessed whether pre-treatment with fluconazole (10 and 20 mg/kg, i.p.) 1 hour prior to ketamine or HNKs (10 mg/kg, i.p.) administration would alter behavioral and neurochemical actions of the drugs in male BALB/cJ mice with a highly anxious phenotype. Extracellular microdialysate levels of glutamate and GABA (Gluext, GABAext) were also measured in the medial prefrontal cortex (mPFC). Pre-treatment with fluconazole altered the pharmacokinetic profile of ketamine, by increasing both plasma and brain levels of ketamine and (R,S)-norketamine, while robustly reducing those of (6)-HNKs. At 24 hours post-injection (t24h), fluconazole prevented the sustained AD-like response of ketamine responses in the forced swim test and splash test, as well as the enhanced cortical GABA levels produced by ketamine. A single (2R,6R)-HNK administration selectively rescued the antidepressant-like activity of ketamine in mice pretreated with fluconazole within 24 hours of treatment. Overall, these findings are consistent with an essential role of (6)-HNK in mediating the sustained antidepressant-like effects of ketamine and suggest potential interactions between pharmacological CYPIs and ketamine during antidepressant treatment in patients.

Future shapes present: autonomous goal-directed and sensory-focused mode switching in a Bayesian allostatic network model
Trade-offs between moving to achieve goals and perceiving the surrounding environment highlight the complexity of continually adapting behaviors. The need to switch between goal-directed and sensory-focused modes, along with the goal emergence phenomenon, challenges conventional optimization frameworks, necessitating heuristic solutions. In this study, we propose a Bayesian recurrent neural network framework for homeostatic behavior adaptation via hierarchical multimodal integration. In it, the meta-goal of minimizing predicted future sensory entropy underpins the dynamic self-organization of future sensorimotor goals and their precision regarding the increasing sensory uncertainty due to unusual physiological conditions. We demonstrated that after learning a hierarchical predictive model of a dynamic environment through random exploration, our Bayesian agent autonomously switched self-organized behavior between goal-directed feeding and sensory-focused resting. It increased feeding before anticipated food shortages, explaining predictive energy regulation (allostasis) in animals. Our modeling framework opens new avenues for studying brain information processing and anchoring continual behavioral adaptations.

Is ChatGPT detrimental to innovation? A field experiment among university students
ChatGPT represents a momentous technological breakthrough whose implications - along with other AI innovations - are yet to fully materialize. This paper is among the first attempts to experimentally test the effect of AI applications (in the form of ChatGPT) on three dependent variables usually assumed to be AI-collaterals: innovation, readiness to exert effort, and risk behaviour. We took advantage of the delayed introduction of ChatGPT in Egypt and conducted a pre-registered field experiment with nearly 100 senior university students at a public university. Over one month during term time, participants were asked to submit three graded essay assignments. In the treatment group, students were asked to write the essays using ChatGPT whereas in the control group, such option was neither mentioned nor allowed (the experiment was fielded before ChatGPT was legally operable in Egypt). One week after all assignments were submitted, the two groups were invited to the lab to play an innovation game (deploying multiple strategies to increase the sales of a hypothetical lemonade stand), a risk game (bomb risk elicitation task), and do a real effort task. The ChatGPT group was significantly less innovative, significantly less risk averse, and exerted less effort (however not statistically significant). Our results point to possible negative effects of AI applications but need further testing and larger samples to be confirmed.

Hearing in categories aids speech streaming at the "cocktail party"
Our perceptual system bins elements of the speech signal into categories to make speech perception manageable. Here, we aimed to test whether hearing speech in categories (as opposed to a continuous/gradient fashion) affords yet another benefit to speech recognition: parsing noisy speech at the "cocktail party." We measured speech recognition in a simulated 3D cocktail party environment. We manipulated task difficulty by varying the number of additional maskers presented at other spatial locations in the horizontal soundfield (1-4 talkers) and via forward vs. time-reversed maskers, promoting more and less informational masking (IM), respectively. In separate tasks, we measured isolated phoneme categorization using two-alternative forced choice (2AFC) and visual analog scaling (VAS) tasks designed to promote more/less categorical hearing and thus test putative links between categorization and real-world speech-in-noise skills. We first show that listeners can only monitor up to [~]3 talkers despite up to 5 in the soundscape and streaming is not related to extended high-frequency hearing thresholds (though QuickSIN scores are). We then confirm speech streaming accuracy and speed decline with additional competing talkers and amidst forward compared to reverse maskers with added IM. Dividing listeners into "discrete" vs. "continuous" categorizers based on their VAS labeling (i.e., whether responses were binary or continuous judgments), we then show the degree of IM experienced at the cocktail party is predicted by their degree of categoricity in phoneme labeling; more discrete listeners are less susceptible to IM than their gradient responding peers. Our results establish a link between speech categorization skills and cocktail party processing, with a categorical (rather than gradient) listening strategy benefiting degraded speech perception. These findings imply figure-ground deficits common in many disorders might arise through a surprisingly simple mechanism: a failure to properly bin sounds into categories.

Human iPSC-derived cell grafts promote functional recovery by molecular interaction with stroke-injured brain
Stroke is a leading cause of disability and death due to the brains limited ability to regenerate damaged neural circuits. To date, stroke patients have only few therapeutic options and are often left with considerable disabilities. Induced pluripotent stem cell (iPSC)-based therapies are emerging as a promising therapeutic approach for stroke recovery. In this study, we demonstrate that local transplantation of good manufacturing practice (GMP)-compatible iPSC-derived neural progenitor cells (NPCs) improve long-term recovery-associated brain tissue responses and reduce neurological deficits after cerebral ischemia in mice. Using in vivo bioluminescence imaging and post-mortem histology, we showed long-term graft survival over the course of five weeks and preferential graft differentiation into mature neurons without signs of pluripotent residuals. Transplantation of NPCs led to a set of recovery-associated tissue responses including increased vascular sprouting and repair, improved blood-brain barrier integrity, reduced microglial activation, and increased neurogenesis compared to littermate control animals receiving sham transplantation. Employing deep learning-assisted behavior analysis, we found that NPC-treated mice displayed improved gait performance and complete fine-motor recovery in the horizontal ladder rung walk, five weeks post-injury. To dissect the molecular graft composition and identify graft-host interactions, single nucleus profiling of the cell transplants and host stroke tissue was performed. We identified graft differentiation preferentially towards GABAergic cells with remaining cells acquiring glutamatergic neuron, astrocyte, and NPC-like phenotypes. Interaction between graft and host transcriptome indicated that GABAergic cell grafts were primarily involved in graft-host communication through the regeneration-associated NRXN, NRG, NCAM and SLIT signalling pathways. In conclusion, our study reveals that transplanted iPSC-derived NPCs primarily differentiate into GABAergic neurons contributing to long-term recovery, and further delineates the regenerative interactions between the graft and the stroke-injured host tissue.

Modulation of the p75NTR during adolescent alcohol exposure prevents cholinergic neuronal atrophy and associated acetylcholine activity and behavioral dysfunction
Binge alcohol consumption during adolescence produces lasting deficits in learning and memory, while also increasing the susceptibility to substance use disorders. The adolescent intermittent ethanol (AIE) rodent model mimics human adolescent binge drinking and has identified the Nucleus Basalis Magnocellularis (NbM) as a key site of pathology. The NbM is a critical regulator of prefrontal cortical (PFC) cholinergic function and attention. The cholinergic phenotype is controlled pro/mature neurotrophin receptor activation. We sought to determine if p75NTR activity contributes to the loss of cholinergic phenotype in AIE by using a p75NTR modulator (LM11A-31) to inhibit prodegenerative signaling during ethanol exposure. Male and female rats underwent 5g/kg ethanol (AIE) or water (CON) exposure following 2-day-on 2-day-off cycles from PND 25-57. A subset of these groups also received a protective dose of LM11A-31 (50mg/kg) during adolescence. Rats were trained on a sustained attention task (SAT) while recording activity with a fluorescent acetylcholine indicator (AChGRAB 3.0). AIE produced learning deficits on the SAT, which were spared with LM11A-31. In addition, mPFC ACh activity was blunted by AIE, which LM11A-31 corrected. Investigation of NbM ChAT+ and TrkA+ neuronal expression found that AIE led to a reduction of ChAT+TrkA+ neurons, which again LM11A-31 protected. Taken together these findings demonstrate the p75NTR activity during AIE treatment is a key regulator of cholinergic degeneration.

Intracranial Mapping of Response Latencies and Task Effects for Spoken Syllable Processing in the Human Brain
Prior lesion, noninvasive-imaging, and intracranial-electroencephalography (iEEG) studies have documented hierarchical, parallel, and distributed characteristics of human speech processing. Yet, there have not been direct, intracranial observations of the latency with which regions outside the temporal lobe respond to speech, or how these responses are impacted by task demands. We leveraged human intracranial recordings via stereo-EEG to measure responses from diverse forebrain sites during (i) passive listening to /bi/ and /pi/ syllables, and (ii) active listening requiring /bi/-versus-/pi/ categorization. We find that neural response latency increases from a few tens of ms in Heschls gyrus (HG) to several tens of ms in superior temporal gyrus (STG), superior temporal sulcus (STS), and early parietal areas, and hundreds of ms in later parietal areas, insula, frontal cortex, hippocampus, and amygdala. These data also suggest parallel flow of speech information dorsally and ventrally, from HG to parietal areas and from HG to STG and STS, respectively. Latency data also reveal areas in parietal cortex, frontal cortex, hippocampus, and amygdala that are not responsive to the stimuli during passive listening but are responsive during categorization. Furthermore, multiple regions--spanning auditory, parietal, frontal, and insular cortices, and hippocampus and amygdala--show greater neural response amplitudes during active versus passive listening (a task-related effect). Overall, these results are consistent with hierarchical processing of speech at a macro level and parallel streams of information flow in temporal and parietal regions. These data also reveal regions where the speech code is stimulus-faithful and those that encode task-relevant representations.

New & NoteworthyWe leverage direct, intracranial electroencephalography recordings to document speech information flow through diverse sites in the human forebrain, including areas where reported electrode coverage has historically been limited. Our data are consistent with hierarchical processing of speech at a macro level and parallel streams of information flow in temporal and parietal regions. They also reveal regions in the auditory pathway where stimulus-faithful speech codes are transformed to behaviorally relevant representations of speech content.

Guardian of Excitability: Multifaceted Role of Galanin in Whole Brain Excitability
Galanin is a neuropeptide, which is critically involved in homeostatic processes like controlling arousal, sleep, and regulation of stress. This extensive range of functions aligns with implications of galanin in diverse pathologies, including anxiety disorders, depression, and epilepsy. Here we investigated the regulatory function of galanin on whole-brain activity in larval zebrafish using wide-field Ca2+ imaging. Combining this with genetic perturbations of galanin signaling and pharmacologically increasing neuronal activity, we are able to probe actions of galanin across the entire brain. Our findings demonstrate that under unperturbed conditions and during epileptic seizures, galanin exerts a sedative influence on the brain, primarily through the galanin receptor 1a (galr1a). However, exposure to acute stressors like pentylenetetrazole (PTZ) compromises galanin's sedative effects, leading to overactivation of the brain and increased seizure occurrence. Interestingly, galanin's impact on seizures appears to be bidirectional, as it can both decrease seizure severity and increase seizure occurrence, potentially through different galanin receptor subtypes. This nuanced interplay between galanin and various physiological processes underscores its significance in modulating stress-related pathways and suggests its potential implications for neurological disorders such as epilepsy. Taken together, our data sheds light on a multifaceted role of galanin, where galanin regulates whole-brain activity but also shapes acute responses to stress.

Temporal Dynamics of Neocortical Development in Organotypic Mouse Cultures: A Comprehensive Analysis
Murine organotypic brain slice cultures have been widely used in neuroscientific research and are offering the opportunity to study neuronal function under normal and disease conditions. Despite the brought application, the mechanisms governing the maturation of immature cortical circuits in vitro are not well understood. In this study, we present a detailed investigation into the development of the neocortex in vitro. Utilizing a holistic approach, we studied organotypic whole-hemisphere brain slice cultures from postnatal mice and tracked the development of the somatosensory area over a five-week period. Our analysis revealed the maturation of passive and active intrinsic properties of pyramidal cells together with their morphology, closely resembling in vivo development. Detailed Multi-electrode array (MEA) electrophysiological assessments and RNA expression profiling demonstrated stable network properties by two weeks in culture, followed by the transition of spontaneous activity towards more complex patterns including high-frequency oscillations. However, weeks 4 and 5 exhibited increased variability and initial signs of neuronal loss, highlighting the importance of considering developmental stages in experimental design. This comprehensive characterization is vital for understanding the temporal dynamics of the neocortical development in vitro, with implications for neuroscientific research methodologies, particularly in the investigation of diseases such as epilepsy and other neurodevelopmental disorders.

Modulation of metastable ensemble dynamics explains optimal coding at moderate arousal in auditory cortex
Performance during perceptual decision-making exhibits an inverted-U relationship with arousal, but the underlying network mechanisms remain unclear. Here, we recorded from auditory cortex (A1) of behaving mice during passive tone presentation, while tracking arousal via pupillometry. We found that tone discriminability in A1 ensembles was optimal at intermediate arousal, revealing a population-level neural correlate of the inverted-U relationship. We explained this arousal-dependent coding using a spiking network model with a clustered architecture. Specifically, we show that optimal stimulus discriminability is achieved near a transition between a multi-attractor phase with metastable cluster dynamics (low arousal) and a single-attractor phase (high arousal). Additional signatures of this transition include arousal-induced reductions of overall neural variability and the extent of stimulus-induced variability quenching, which we observed in the empirical data. Our results elucidate computational principles underlying interactions between pupil-linked arousal, sensory processing, and neural variability, and suggest a role for phase transitions in explaining nonlinear modulations of cortical computations.

Missense mutation in the activation segment of the kinase CK2 models Okur Chung neurodevelopmental disorder and alters the hippocampal glutamatergic synapse
Exome sequencing has enabled the identification of causative genes of monogenic forms of autism, amongst them, in 2016, CSNK2A1, the gene encoding the catalytic subunit of the kinase CK2, linking this kinase to Okur-Chung Neurodevelopmental Syndrome (OCNDS), a newly described neurodevelopmental condition with many symptoms resembling those of autism spectrum disorder.

Thus far, no preclinical model of this condition exists. Here we describe a knockin mouse model that harbors the K198R mutation in the activation segment of the kinase. This region is a mutational hotspot, representing one-third of patients. These mice exhibit behavioral phenotypes that mirror patient symptoms. Homozygous knock-in (KI) mice die mid-gestation while heterozygous KI mice are born at half of the expected mendelian ratio and are smaller in weight and size than wildtype littermates. Heterozygous KI mice showed alterations in cognition and memory-assessing paradigms, enhanced stereotypies, altered circadian activity patterns, and nesting behavior. Phosphoproteome analysis from brain tissue revealed alterations in the phosphorylation status of major pre- and postsynaptic proteins of heterozygous KI mice. In congruence, we detect reduced synaptic maturation in hippocampal neurons and attenuated long-term potentiation in the hippocampus of KI mice. Taken together, K198R KI mice exhibit significant face validity, presenting ASD-relevant phenotypes, synaptic deficits and alterations in synaptic plasticity, all of which strongly validate this line as a mouse model of OCNDS.

Adolescent Seizure Impacts Oligodendrocyte Development, Neuronal-Glial Circuit Formation, and Myelination
Myelin sheaths, formed by oligodendrocyte cells in the CNS, are vital for rapid conduction of electrical signals down neuronal axons. Oligodendrocyte progenitors differentiate and myelinate axons during development and following demyelinating injury. However, the mechanisms that drive the timing and specificity of developmental myelination are not well understood. It is known that oligodendrocyte progenitors receive synapses from neurons, providing a potential mechanism for neuronal-glial communication. We have previously shown that changing neuronal activity affects the proliferation of oligodendrocyte cells and neuron to OPC connections. We hypothesized that OPC proliferation and differentiation would be affected by pathological neuronal activity during adolescent development, when developmental myelination is occurring, and that this would also impact neuron to OPC connectivity and myelination. We used kainic acid to induce a seizure, then analyzed changes in the rate of OPC proliferation and differentiation five days later in the cerebral cortex, corpus callosum, and hippocampus. We found that OPC proliferation increased, the overall numbers of OPCs increased, and the number of mature oligodendrocytes decreased. We measured changes in the myelination to determine whether seizure activity directly affected myelination rate in adolescent development, and found decreased myelin in the cerebral cortex, corpus callosum, and hippocampus. We used viral monosynaptic circuit tracing to determine whether connections between neurons and OPCs were affected by seizure activity, and found a decrease in neuron to OPC connections in seizure mice compared to controls. Finally, we measured changes in the presence of kir4.1 potassium channels in OPCs, an important regulator of OPC membrane potential as well as an ion channel important for myelination, and found that there was a decrease in the number of potassium channels on OPCs after adolescent seizure. These findings provide insight into the response of the adolescent brain to seizure activity, as well as how seizures affect neuronal glial connections, OPC development and myelin formation, with the goal of understanding how these mechanisms may be important for treatment of demyelination after seizure and in epilepsy.

Three-photon excited fluorescence microscopy enables imaging of blood flow, neural structure and inflammatory response deep into mouse spinal cord in vivo
Nonlinear optical microscopy enables non-invasive imaging in scattering samples with cellular resolution. The spinal cord connects the brain with the periphery and governs fundamental behaviors such as locomotion and somatosensation. Because of dense myelination on the dorsal surface, imaging to the spinal grey matter is challenging, even with two-photon microscopy. Here we show that three-photon excited fluorescence (3PEF) microscopy enables multicolor imaging at depths of up to ~550 m into the mouse spinal cord, in vivo. We quantified blood flow across vessel types along the spinal vascular network. We then followed the response of neurites and microglia after occlusion of a surface venule, where we observed depth-dependent structural changes in neurites and interactions of perivascular microglia with vessel branches upstream from the clot. This work establishes that 3PEF imaging enables studies of functional dynamics and cell type interactions in the top 550 m of the murine spinal cord, in vivo.