bioRxiv Subject Collection: Neuroscience's Journal

Optogenetic inhibition of light-captured alcohol-taking striatal engrams facilitates extinction and suppresses reinstatement
Background: Alcohol use disorder (AUD) is a complex condition, and it remains unclear which specific neuronal substrates mediate alcohol-seeking and -taking behaviors. Engram cells and their related ensembles, which encode learning and memory, may play a role in this process. We aimed to assess the precise neural substrates underlying alcohol-seeking and -taking behaviors and determine how they may affect one another. Methods: Using FLiCRE (Fast Light and Calcium-Regulated Expression; a newly developed technique which permits the trapping of acutely activated neuronal ensembles) and operant-self administration (OSA), we tagged striatal neurons activated during alcohol-taking behaviors. We used FLiCRE to express an inhibitory halorhodopsin in alcohol-taking neurons, permitting loss-of-function manipulations. Results: We found that the inhibition of OSA-tagged alcohol-taking neurons decreased both alcohol-seeking and -taking behaviors in future OSA trials. In addition, optogenetic inhibition of these OSA-tagged alcohol-taking neurons during extinction training facilitated the extinction of alcohol-seeking behaviors. Furthermore, inhibition of these OSA-tagged alcohol-taking neurons suppressed the reinstatement of alcohol-seeking behaviors, but, interestingly, it did not significantly suppress alcohol-taking behaviors during reinstatement. Conclusions: Our findings suggest that alcohol-taking neurons are crucial for future alcohol-seeking behaviors during extinction and reinstatement. These results may help in the development of new therapeutic approaches to enhance extinction and suppress relapse in individuals with AUD.

Mechanosensory representation of wing deformations
Efficient representation of structural deformations is crucial for monitoring the instantaneous state of biological structures. Insects' ability to encode wing deformations during flight demonstrates a general morphological computing principle applicable across sensory systems in nature as well as engineered systems. To characterize how relevant features are encoded, we measured and modelled displacement and strain across dragonfly wing surfaces in tethered and free flight. Functional interpretations were supported by neuroanatomical maps, and ablation and perturbation experiments. We find that signal redundancy is reduced by non-random sensor distributions and that morphology limits the stimulus space such that sensory systems can monitor natural states with few sensors. Deviations from the natural states are detected by a flexible population of additional sensors with many distinguishable activation patterns.

An Amygdalar-Vagal-Glandular Circuit Controls the Intestinal Microbiome
Psychological states can regulate intestinal mucosal immunity by altering the gut microbiome. However, the link between the brain and microbiome composition remains elusive. We show that Brunner's glands in the duodenal submucosa couple brain activity to intestinal bacterial homeostasis. Brunner's glands mediated the enrichment of gut probiotic species in response to stimulation of abdominal vagal fibers. Cell-specific ablation of the glands triggered a transmissible dysbiosis associated with an immunodeficiency syndrome that led to mortality upon gut infection with pathogens. The syndrome could be largely prevented by oral or intra-intestinal administration of probiotics. In the forebrain, we identified a vagally-mediated, polysynaptic circuit connecting the glands of Brunner to the central nucleus of the amygdala. Intra-vital imaging revealed that excitation of central amygdala neurons activated Brunner's glands and promoted the growth of probiotic populations. Our findings unveil a vagal-glandular neuroimmune circuitry that may be targeted for the modulation of the gut microbiome.

Endogenous tau released from human ReNCell VM cultures by neuronal activity is phosphorylated at multiple sites
Tau is an intracellular protein but also known to be released into the extracellular fluid. Tau release mechanisms have drawn intense attention as these are known to play a key role in Alzheimer's disease (AD) pathology. However, tau can also be released under physiological conditions although its physiological function and release mechanisms have been poorly characterized, especially in human neuronal cells. We investigated endogenous tau release in ReNCell VM, a human neuroprogenitor cell line, under physiological conditions and found that tau is spontaneously released from cells. To study activity-dependent release of endogenous tau, human ReNCell VM culture was stimulated by 100M AMPA or 50mM KCl for one-hour, tau was actively released to the culture medium. The released tau was highly phosphorylated at nine phosphorylation sites (pSites) detected by phospho-specific tau antibodies including AT270 (T175/T181), AT8 (S202/T205), AT100 (T212/S214), AT180 (T231), and PHF-1 (S396/S404), showing that these pSites are important for activity-dependent tau release from human ReNCell VM. Intracellular tau showed various phosphorylation status across these sites, with AT270 and PHF-1 highly phosphorylated while AT8 and AT180 were minimally phosphorylated, suggesting that AT8 and AT180 pSites exhibit a propensity for secretion rather than being retained intracellularly. This activity-dependent tau release was significantly decreased by inhibition of GSK-3{beta}, demonstrating that GSK3{beta}-dependent phosphorylation of tau plays an important role in its release by neuronal activity. In this study, we showed that ReNCell VM serves as a valuable model for studying endogenous physiological tau release. Further, ReNCell model can be also used to study pathological release of human tau that will contribute to our understanding of the progression of AD and related dementias.

Facial expressions in mice reveal latent cognitive variables and their neural correlates
Brain activity controls adaptive behavior but also drives unintentional incidental movements. Such movements could thus potentially be used to read out internal cognitive variables also neurally computed. Establishing this, however, would require ruling out that incidental movements reflect cognition only because they are coupled with task-related responses through the biomechanics of the body. We addressed this issue in a foraging task for mice where multiple decision variables are simultaneously encoded even if, at any given time, only one of them is used. We found that characteristic features of the face simultaneously encode not only the currently used decision variables, but also independent and unexpressed ones, and we show that these features partially originate from neural activity in the secondary motor cortex. Our results suggest that the face reflects ongoing computations above and beyond those related to task demands, demonstrating the ability of noninvasive monitoring to expose otherwise latent cognitive states.

Inner limiting Membrane Peel Extends In vivo Calcium Imaging of Retinal Ganglion Cell Activity Beyond the Fovea in Non-Human Primate
High resolution retinal imaging paired with intravitreal injection of a viral vector coding for the calcium indicator GCaMP has enabled visualization of activity dependent calcium changes in retinal ganglion cells (RGCs) at single cell resolution in the living eye. The inner limiting membrane (ILM) is a barrier for viral vectors, restricting transduction to a ring of RGCs serving the fovea in both humans and non-human primates (NHP). We evaluate peeling the ILM prior to intravitreal injection as a strategy to expand calcium imaging beyond the fovea in the NHP eye in vivo. Five Macaca fascicularis eyes (age 3-10y; n=3 individuals; 2M, 1F) underwent vitrectomy and 5 to 6-disc diameter ILM peel centered on the fovea prior to intravitreal delivery of 7m8:SNCG:GCaMP8s. Calcium responses from RGCs were recorded using a fluorescence adaptive optics scanning laser ophthalmoscope. In all eyes GCaMP was expressed throughout the peeled area, representing a mean 8-fold enlargement in area of expression relative to a control eye. Calcium recordings were obtained up to 11 degrees from the foveal center. RGC responses were comparable to the fellow control eye and showed no significant decrease over the 6 months post ILM peel, suggesting that RGC function was not compromised by the surgical procedure. In addition, we demonstrate that activity can be recorded directly from the retinal nerve fiber layer. This approach will be valuable for a range of applications in visual neuroscience including pre-clinical evaluation of retinal function, detecting vision loss, and assessing the impact of therapeutic interventions.

Generalized cue reactivity in dopamine neurons after opioids
Cue reactivity is the maladaptive neurobiological and behavioral response upon exposure to drug cues and is a major driver of relapse. The leading hypothesis is that dopamine release by addictive drugs represents a persistently positive reward prediction error that causes runaway enhancement of dopamine responses to drug cues, leading to their pathological overvaluation compared to non-drug reward alternatives. However, this hypothesis has not been directly tested. Here we developed Pavlovian and operant procedures to measure firing responses, within the same dopamine neurons, to drug versus natural reward cues, which we found to be similarly enhanced compared to cues predicting natural rewards in drug-naive controls. This enhancement was associated with increased behavioral reactivity to the drug cue, suggesting that dopamine release is still critical to cue reactivity, albeit not as previously hypothesized. These results challenge the prevailing hypothesis of cue reactivity, warranting new models of dopaminergic function in drug addiction, and provide critical insights into the neurobiology of cue reactivity with potential implications for relapse prevention.

AgRP neurons mediate activity-dependent development of oxytocin connectivity and autonomic regulation
During postnatal life, the adipocyte-derived hormone leptin is required for normal targeting of neural inputs to the paraventricular nucleus of the hypothalamus (PVH) and impacts the activity of neurons containing agouti-related peptide (AgRP) in the arcuate nucleus of the hypothalamus. Activity-dependent developmental mechanisms are known to play a defining role during postnatal organization of neural circuits, but whether leptin-mediated postnatal neuronal activity specifies neural projections to the PVH or impacts downstream connectivity is largely unexplored. Here, we blocked neuronal activity of AgRP neurons during a discrete postnatal period and evaluated development of AgRP inputs to defined regions in the PVH, as well as descending projections from PVH oxytocin neurons to the dorsal vagal complex (DVC) and assessed their dependence on leptin or postnatal AgRP neuronal activity. In leptin-deficient mice, AgRP inputs to PVH neurons were significantly reduced, as well as oxytocin-specific neuronal targeting by AgRP. Moreover, downstream oxytocin projections from the PVH to the DVC were also impaired, despite the lack of leptin receptors on PVH oxytocin neurons. Blocking AgRP activity specifically during early postnatal life reduced the density of AgRP inputs to the PVH, as well as the density of projections from PVH oxytocin neurons to the DVC, and these innervation deficits were associated with dysregulated autonomic function. These findings suggest that postnatal targeting of descending PVH oxytocin projections to the DVC requires leptin-mediated AgRP neuronal activity, and represents a novel activity-dependent mechanism for hypothalamic specification of metabolic circuitry, with consequences for autonomic regulation.

A myelinic channel system for organelle transport to the glial-axonal junction
Myelin sheaths comprise compacted layers of oligodendroglial membrane wrapped spirally around axons. Each sheath, if imagined unwrapped, has a cytoplasm-filled space at its perimeter, linking it to the oligodendrocyte soma via a short process. By electron microscopy (EM), this space, which we term the myelinic channel system contains microtubules and membranous organelles, but whether these are remnants of development or serve a function is unknown. Performing live imaging of myelinating oligodendrocytes expressing fluorescent reporters, we found that the myelinic channel system serves microtubule-dependent organelle transport. Further, the intra-myelinic movement of peroxisomes was modulated by neuronal electrical activity in these mixed neural cell cultures. Loss of oligodendroglial Kif21b or CNP in vivo led to apparent stasis of myelin organelles and secondary axon pathology. This suggests that oligodendrocytes require motor transport in the myelinic channel system to maintain axonal integrity.

Tissue resident memory CD8+ T cells are present but not critical for demyelination and neurodegeneration in a mouse model of multiple system atrophy.
Multiple system atrophy (MSA) is rare, fast progressing, and fatal synucleinopathy with alpha-synuclein (-syn) inclusions located within oligodendroglia called glial cytoplasmic inclusions (GCI). Along with GCI pathology there is severe demyelination, neurodegeneration, and neuroinflammation. In post-mortem tissue, there is significant infiltration of CD8+ T cells into the brain parenchyma, however their role in disease progression is unknown. To determine the role of CD8+ T cells, a modified AAV, Olig001-SYN, was used to selectively overexpress -syn in oligodendrocytes modeling MSA in mice. Four weeks post transduction, we observed significant CD8+ T cell infiltration into the striatum of Olig001-SYN transduced mice recapitulating the CD8+ T cell infiltration observed in post-mortem tissue. To understand the role of CD8+ T cells, a CD8 knockout mice were transduced with Olig001-SYN. Six months post transduction into a mouse lacking CD8+ T cells, demyelination and neurodegeneration were unchanged. Four weeks post transduction, neuroinflammation and demyelination were enhanced in CD8 knockout mice compared to wild type controls. Applying unbiased spectral flow cytometry, CD103+, CD69+, CD44+, CXCR6+, CD8+ T cells were identified when -syn was present in oligodendrocytes, suggesting the presence of tissue resident memory CD8+ T (Trm) cells during MSA disease progression. This study indicates that CD8+ T cells are not critical in driving MSA pathology but are needed to modulate the neuroinflammation and demyelination response.

Spatial and directional tuning of serial dependence for tracking eye movements
An attractive influence of past sensory experience on current behaviour has been observed in many domains, such as for perceptual decisions and motor responses. However, it is unclear what sort of information is integrated across trials, and the limits of this integration, especially for oculomotor behavior. Here we provide a detailed and systematic investigation of the spatial and directional tuning of serial dependence for oculomotor tracking. In a series of experiments, we measured oculomotor responses to sequences of movements: the first movement (the prior) could move at different velocities (5 or 15 deg/s), and could additionally vary in its spatial location or direction relative to the following movement. The second movement (the probe) always moved at the same velocity (10 deg/s) and was constant across all experiments. We observed that eye velocity for the probe movement was faster when following the fast prior compared to following the slow prior, replicating attractive serial dependence. Importantly, this effect stayed consistent for distances of up to 30 deg between probe and prior, strongly suggesting a retinotopic coordinate frame. When we manipulated the direction of the prior, we observed that the strength of the serial dependence on eye velocity as well as eye direction was modulated by the relative angle between prior and probe. We observed stronger serial dependence for prior directions more similar to the probe direction. The strength of the effect on eye velocity and eye direction was correlated, suggesting a shared mechanism controlling these effects. Across all experiments, we observed that even when the prior moved in the opposite direction to the probe, there was a residual attractive effect. This suggests that serial dependence for oculomotor tracking consists of two components, one retinotopic, direction-tuned component and one more general component that is not direction-specific.

Synthesising robot behaviour through reinforcement learning for homeostasis
Homeostasis is a fundamental property for the survival of animals. Computational reinforcement learning provides a theoretically sound framework for learning autonomous agents. However, the definition of a unified motivational signal (i.e., reward) for integrated survival behaviours has been largely underexplored. Here, we present a novel neuroscience-inspired algorithm for synthesising robot survival behaviour without the need for complicated reward design and external feedback. Our agent, the Embodied Neural Homeostat, was trained solely with feedback generated by its internal physical state and optimised its behaviour to stabilise these internal states: homeostasis. To demonstrate the effectiveness of our concept, we trained the agent in a simulated mechano-thermal environment and tested it in a real robot. We observed the synthesis of integrated behaviours, including walking, navigating to food, resting to cool down the motors, and shivering to warm up the motors, through the joint optimisation for thermal and energy homeostasis. The Embodied Neural Homeostat successfully achieved homeostasis-based integrated behaviour synthesis, which has not previously been accomplished at the motor control level. This demonstrates that homeostasis can be a motivating principle for integrated behaviour generation in robots and can also elucidate the behavioural principles of living organisms.

Chemogenetic activation of astrocytes modulates sleep/wakefulness states in a brain region-dependent manner
Study objectives: Astrocytes change their intracellular calcium (Ca2+) concentration during sleep/wakefulness states in mice. Furthermore, the Ca2+ dynamics in astrocytes vary depending on the brain region. However, whether alterations in intracellular Ca2+ concentration in astrocytes can affect sleep/wakefulness states and cortical oscillations in a brain region-dependent manner remain unclear. Methods: The Ca2+ concentration in astrocytes was artificially increased using chemogenetics in mice. Astrocytes in the hippocampus and pons, which are 2 brain regions previously classified into different clusters based on their Ca2+ dynamics during sleep/wakefulness, were focused on to compare whether there are differences in the effects of astrocytes from different brain regions. Results: The activation of astrocytes in the hippocampus significantly decreased the total time of wakefulness and increased the total time of sleep. This had minimal effects on cortical oscillations in all sleep/wakefulness states. On the other hand, the activation of astrocytes in the pons substantially suppressed rapid eye movement (REM) sleep in association with a decreased number of REM episodes, indicating strong inhibition of REM onset. Regarding cortical oscillations, the delta wave component during non-REM sleep was significantly enhanced. Conclusions: These results suggest that astrocytes modulate sleep/wakefulness states and cortical oscillations. Furthermore, the role of astrocytes in sleep/wakefulness states appears to vary among brain regions.

Effects of Tasks on Functional Brain Connectivity Derived from Inter-Individual Correlations: Insights from Regional Homogeneity of Functional MRI Data
Research on brain functional connectivity often relies on intra-individual moment-to-moment correlations of functional brain activity, typically using techniques like functional MRI (fMRI). Inter-individual correlations are also employed on data from fMRI and positron emission tomography (PET). Many past studies have not specified tasks for participants, keeping them in an implicit "resting" condition. This lack of task specificity raises questions about how different tasks impact inter-individual correlation estimates. In our analysis of fMRI data from 100 unrelated participants, scanned during seven task conditions and in a resting state, we calculated Regional Homogeneity (ReHo) for each task as a regional measure of brain functions. We found that changes in ReHo due to different tasks were relatively small compared with the variations across brain regions. Cross-region variations of ReHo were highly correlated between different tasks. Similarly, whole-brain inter-individual correlation patterns were remarkably consistent across the tasks, showing correlations greater than 0.78. Changes in inter-individual correlations between tasks were primarily driven by connectivity in the visual, somatomotor, default mode network, and the interactions between them. The subtle yet statistically significant differences in functional connectivity may be linked to specific brain regions associated with the studied tasks. Future studies should consider task design when exploring inter-individual connectivity in specific brain systems.

Identification of cortical targets for modulating function supported by the human hippocampal network
Background: Individualized transcranial magnetic stimulation (TMS) targeting using functional connectivity analysis of functional magnetic resonance imaging (fMRI) has been demonstrated to be advantageous in inducing neuroplasticity. However, how this approach can benefit modulating the episodic memory function supported by the hippocampal network remains elusive. Objective: We use the resting-state fMRI data from a large cohort to reveal tentative TMS targets at cortical regions within the hippocampal network. Methods: Functional MRI from 1,133 individuals in the Human Connectome Project was used to analyze the hippocampal network using seed-based functional connectivity. Results: Using a weighted sum of time series at the cortex, we identified the average centroids of individualized targets at the medial prefrontal cortex (mPFC) and posterior parietal cortices (PPC) at (-10, 49, 7) and (-40, -67, 30) in the left hemisphere, respectively. The mPFC and PPC coordinate at the right hemispheres are (11, 51, 6) and (48, -59, 24) in the right hemisphere, respectively. These coordinates can be reliably identified (>90% of individuals). Conclusion: Our results suggest candidate TMS target coordinates to modulate the hippocampal function.

Different Trajectories of Functional Connectivity Captured with Gamma-Event Coupling and Broadband Measures of EEG in the Rat Fluid Percussion Injury Model.
Functional connectivity (FC) after TBI is affected by an altered excitatory-inhibitory balance due to neuronal dysfunction, and the mechanistic changes observed could be reflected differently by contrasting methods. Local gamma event coupling FC (GEC-FC) is believed to represent multiunit fluctuations due to inhibitory dysfunction, and we hypothesized that FC derived from widespread, broadband amplitude signal (BBA-FC) would be different, reflecting broader mechanisms of functional disconnection. We tested this during sleep and active periods defined by high delta and theta EEG activity, respectively, at 1,7 and 28d after rat fluid-percussion-injury (FPI) or sham injury (n=6/group) using 10 indwelling, bilateral cortical and hippocampal electrodes. We also measured seizure and high-frequency oscillatory activity (HFOs) as markers of electrophysiological burden. BBA-FC analysis showed early hyperconnectivity constrained to ipsilateral sensory-cortex-to-CA1-hippocampus that transformed to mainly ipsilateral FC deficits by 28d compared to shams. These changes were conserved over active epochs, except at 28d when there were no differences to shams. In comparison, GEC-FC analysis showed large regions of hyperconnectivity early after injury within similar ipsilateral and intrahemispheric networks. GEC-FC weakened with time, but hyperconnectivity persisted at 28d compared to sham. Edge- and global connectivity measures revealed injury-related differences across time in GEC-FC as compared to BBA-FC, demonstrating greater sensitivity to FC changes post-injury. There was no significant association between sleep fragmentation, HFOs, or seizures with FC changes. The within-animal, spatial-temporal differences in BBA-FC and GEC-FC after injury may represent different mechanisms driving FC changes as a result of primary disconnection and interneuron loss.

Ecological fNIRS in mobile children: Using short separation channels to correct for systemic contamination during naturalistic neuroimaging.
Significance The advances and the miniaturization in functional Near Infrared Spectroscopy (fNIRS) instrumentation offers the potential to move the classical laboratory-based cognitive neuroscience investigations into more naturalistic settings. Wearable and mobile fNIRS devices also provide a novel child-friendly means to image functional brain activity in freely moving toddlers and preschoolers. Measuring brain activity in more ecologically valid settings with fNIRS presents additional challenges, such as the increased impact of physiological interferences. One of the most popular methods to minimize such interferences is to regress out short separation channels from the long separation channels (i.e., superficial signal regression or SSR). Whilst this has been extensively investigated in adults, little is known about the impact of systemic changes on the fNIRS signals recorded in children in either classical or novel naturalistic experiments. Aim We aim to investigate if extracerebral physiological changes occur in toddlers and preschoolers, and whether SSR can help minimize these interferences. Approach We collected fNIRS data from 3-to-7 years olds during a conventional computerized static task and in a dynamic naturalistic task in an immersive virtual reality (VR) continuous automatic virtual environment (CAVE). Results Our results show that superficial signal contamination data is present in both young children as in adults. Importantly, we find that SSR helps in improving the localization of functional brain activity, both in the computerized task and, to a larger extent, in the dynamic VR task. Conclusions Following from these results, we formulate suggestions to advance the field of developmental neuroimaging with fNIRS, particularly in ecological settings.

Nonlinear spatial integration allows the retina to detect the sign of defocus in natural scenes
Eye growth is regulated by the visual input. Many studies suggest that the retina can detect if a visual image is focused in front or behind the back of the eye, and modulate eye growth to bring it back to focus. How can the retina distinguish between these two types of defocus? Here we simulated how eye optics transform natural images and recorded how the isolated retina responds to different types of simulated defocus. We found that some ganglion cell types could distinguish between an image focussed in front or behind the retina, by estimating spatial contrast. Aberrations in the eye optics made spatial contrast, but not luminance, a reliable cue to distinguish these two types of defocus. Our results suggest a mechanism for how the retina can estimate the sign of defocus and provide an explanation for several results aiming at mitigating strong myopia by slowing down eye growth.

Cognitive behavioral phenotyping of DSCAM heterozygosity as a model for autism spectrum disorder
It is estimated that 1 in 36 children are affected by autism spectrum disorder (ASD) in the United States, which is nearly a twofold increase from a decade ago. Recent genetic studies have identified de novo loss-of-function (dnLoF) mutations in the Down Syndrome Cell Adhesion Molecule (DSCAM) as a strong risk factor for ASD. Previous research has shown that DSCAM ablation confers social interaction deficits and perseverative behaviors in mouse models. However, it remains unknown to what extent DSCAM underexpression captures the full range of behaviors, specifically cognitive phenotypes, presented in ASD. Here, we conducted a comprehensive cognitive behavioral phenotyping which revealed that loss of one copy of DSCAM, as in the DSCAM2J+/- mice, displayed hyperactivity, increased anxiety, and motor coordination impairments. Additionally, hippocampal-dependent learning and memory was affected, including working memory, long-term memory, and contextual fear learning. Interestingly, implicit learning processes remained intact. Therefore, DSCAM LoF produces autistic-like behaviors that are similar to human cases of ASD. These findings further support a role for DSCAM dnLoF mutations in ASD and suggest DSCAM2J+/- as a suitable model for ASD research.