bioRxiv Subject Collection: Neuroscience's Journal

miRNA-mediated control of gephyrin synthesis drives sustained inhibitory synaptic plasticity
Activity-dependent protein synthesis is crucial for many long-lasting forms of synaptic plasticity. However, our understanding of the translational mechanisms controlling inhibitory synapses is limited. One distinct form of inhibitory long-term potentiation (iLTP) enhances postsynaptic clusters of GABAARs and the primary inhibitory scaffold, gephyrin, to promote sustained synaptic strengthening. While we previously found that persistent iLTP requires mRNA translation, the precise mechanisms controlling gephyrin translation during this process remain unknown. Here, we identify miR153 as a novel regulator of Gphn mRNA translation which controls gephyrin protein levels and synaptic clustering, ultimately impacting GABAergic synaptic structure and function. We find that iLTP induction downregulates miR153, reversing its translational suppression of Gphn mRNA and allowing for increased de novo gephyrin protein synthesis and synaptic clustering during iLTP. Finally, we find that reduced miR153 expression during iLTP is driven by an excitation-transcription coupling pathway involving calcineurin, NFAT and HDACs, which also controls the miRNA-dependent upregulation of GABAARs. Overall, this work delineates a miRNA-dependent post-transcriptional mechanism that controls the expression of the key synaptic scaffold, gephyrin, and may converge with parallel miRNA pathways to coordinate gene upregulation to maintain inhibitory synaptic plasticity.

Role of Ribeye PXDLS/T-binding cleft in normal synaptic ribbon function
Non-spiking sensory hair cells of the auditory and vestibular systems encode a dynamic range of graded signals with high fidelity by vesicle exocytosis at ribbon synapses. Ribeye, the most abundant protein in the synaptic ribbon, is composed of a unique A domain specific for ribbons and a B-domain nearly identical to the transcriptional corepressor CtBP2. CTBP2 and the B-domain of Ribeye contain a surface cleft that binds to proteins harboring a PXDLS/T peptide motif. Little is known about the importance of this binding site in synaptic function. Piccolo has a well-conserved PVDLT motif and we find that overexpressed Ribeye exhibits striking co-localization with Piccolo in INS-cells, while two separate mutants containing mutations in PXDLS/T-binding region, fail to co-localize with Piccolo. Similarly, co-transfected Ribeye and a piccolo fragment containing the PVDLT region co-localize in HEK cells. Expression of wild-type Ribeye-YFP in zebrafish neuromast hair cells returns electron densities to ribbon structures and mostly rescued normal synaptic transmission and morphological phenotypes in a mutant zebrafish lacking most Ribeye. By contrast, Ribeye-YFP harboring a mutation in the PXDLS/T-binding cleft resulted in ectopic electron dense aggregates that did not collect vesicles and the persistence of ribbons lacking electron densities. Furthermore, overexpression failed to return capacitance responses to normal levels. These results point toward a role for the PXDLS/T-binding cleft in the recruitment of Ribeye to ribbons and in normal synaptic function.

Organelle phenotyping and multi-dimensional microscopy identify C1q as a novel regulator of microglial function.
Microglia, the immune cells of the central nervous system (CNS), are incredibly dynamic and heterogenous cells. While single cell RNA sequencing has become the conventional methodology for evaluating microglial state, transcriptomics do not provide insight into functional changes. Here, we propose a novel organelle phenotyping approach where we treat live human induced pluripotent stem cell-derived microglia (iMGL) with organelle dyes (mitochondria, lipids, lysosomes) and acquire data by live-cell spectral microscopy. Dimensionality reduction techniques and unbiased cluster identification allow for recognition of microglial subpopulations based in organelle function. We validate this methodology using lipopolysaccharide (LPS) and IL-10 treatment to polarize iMGL to an inflammatory and anti-inflammatory state, respectively, and then apply it to identify a novel regulator of iMGL function, complement protein C1q. C1q is traditionally known as the initiator of the complement cascade, but here we use organelle phenotyping to identify a role for C1q in regulating iMGL fatty acid storage and mitochondria membrane potential. Follow up evaluation of microglia with more traditional read outs of activation state confirm that C1q drives an increase in microglia pro-inflammatory cytokine production and migration, while suppressing microglial proliferation. These data together validate the use of a novel organelle phenotyping approach and enable better mechanism investigation of molecular regulators of microglial state, such as C1q.

Variant Mapping Application: Customize And Annotate Figures Of Voltage-Gated Sodium Channel
Voltage-gated sodium ion channels allow for the initiation and transmission of action potentials. There is a high interest in research and drug development to selectively target these ion channels to treat epilepsy and other disorders such as pain. Scientific literature and presentations often incorporate maps of these integral membrane proteins with markers indicating gene mutations to highlight genotype/phenotype correlations. There is a need for automated tools to create high quality figures with mutation (variant) locations displayed on these channel maps. This manuscript introduces a simple application to create visualization for mutations on alpha voltage-gated sodium channels, created using the D3.js library. The application allows for mapping of variant sequences, as well as important properties like the type of variant and the phenotypes linked to the variant. It also allows for customizability and the production of high-quality images for publication. This application and code base can further be extrapolated to other ion channels as well.

Senolytic Intervention Improves Cognition, Metabolism, and Adiposity in Female APPNL-F/NL-F Mice.
INTRODUCTION: Senescent cells accumulate throughout the body and brain contributing to unhealthy aging and Alzheimer's disease (AD). We hypothesized that senolytic intervention would alleviate cellular senescence thereby improving spatial memory in APPNL-F/NL-F mice. METHODS: Male and female APPNL-F/NL-F mice were treated monthly with vehicle, 5 mg/kg Dasitinib (D) + 50 mg/kg Quercetin (Q), or 100 mg/kg Fisetin. Blood glucose levels, energy metabolism, spatial memory, and senescent cell markers were assayed. RESULTS: D+Q treatment in female APPNL-F/NL-F mice increased oxygen consumption and energy expenditure resulting in decreased body mass. White adipose tissue content was decreased along with senescence markers, SASP, blood glucose, and plasma insulin and triglycerides. Hippocampal senescence markers and SASP were reduced along with soluble and insoluble A{beta}42 and SA-{beta}-gal activity leading to improved spatial memory. DISCUSSION: Considering women have a greater risk of dementia, identifying senotherapeutics appropriate for sex and disease stage is necessary for personalized medicine.

Single cell discovery of m6A RNA modifications in the hippocampus
N6-methyladenosine (m6A) is a prevalent and highly regulated RNA modification essential for RNA metabolism and normal brain function. It is particularly important in the hippocampus, where m6A is implicated in neurogenesis and learning. Although extensively studied, its presence in specific cell types remain poorly understood. We investigated m6A in the hippocampus at the single cell level, revealing a comprehensive landscape of m6A modifications within individual cells. Our data also identifies transcripts which have high m6A density and are associated with brain diseases. Our data suggests that m6A containing transcripts might be of particular importance for Camk2a neurons. Overall, this work provides new insights into the molecular mechanisms underlying hippocampal physiology and lays the foundation for future studies investigating the dynamic nature of m6A RNA methylation in the healthy and diseased brain.

Geometry and dynamics of representations in a precisely balanced memory network related to olfactory cortex
Biological memory networks are thought to store information in the synaptic connectivity between assemblies of neurons. Recent models suggest that these assemblies contain both excitatory and inhibitory neurons (E/I assemblies), resulting in co-tuning and precise balance of excitation and inhibition. To understand the computational consequences of E/I assemblies under biologically realistic constraints we created a spiking network model based on experimental data from telencephalic area Dp of adult zebrafish, a precisely balanced recurrent network homologous to piriform cortex. We found that E/I assemblies stabilized firing rate distributions compared to networks with excitatory assemblies and global inhibition. Unlike classical memory models, networks with E/I assemblies did not show discrete attractor dynamics. Rather, responses to learned inputs were locally constrained onto manifolds that "focused" activity into neuronal subspaces. The covariance structure of these manifolds supported pattern classification when information was retrieved from selected neuronal subsets. Networks with E/I assemblies therefore transformed the geometry of neuronal coding space, resulting in continuous representations that reflected both relatedness of inputs and an individual's experience. Such continuous internal representations enable fast pattern classification, can support continual learning, and may provide a basis for higher-order learning and cognitive computations.

Neurophysiological mechanisms underlying post-stroke deficits in contralesional perceptual processing
Slowed responding to sensory inputs presented in contralesional space is pervasive following unilateral cerebral stroke, but the causal neurophysiological pathway by which this occurs remains unclear. To this end, here we leverage a perceptual decision-making framework to disambiguate information processing stages between sensation and action in 30 unilateral stroke patients (18 right hemisphere, 12 left hemisphere) and 27 neurologically healthy adults. By recording neural activity using electroencephalography (EEG) during task performance, we show that the relationship between strokes in either hemisphere and slowed contralesional response times is sequentially mediated by weaker target selection signals in the contralateral hemisphere (the N2c ERP), and subsequently delayed evidence accumulation signals (the centroparietal positivity). Notably, asymmetries in CPP and response times across hemispheres are associated with everyday functioning. Together, these data suggest a plausible neurophysiological pathway by which post-stroke contralesional slowing arises and highlight the utility of neurophysiological assessments for tracking clinically relevant behaviour.

Involvement of FKBP5, but not of stress, in alcohol memory reconsolidation
Relapse is a fundamental challenge in drug addiction, often evoked by exposure to drug-associated cues. Upon retrieval, memories become temporarily labile before re-stabilizing in a process termed reconsolidation. Therefore, targeting the reconsolidation process offers a therapeutic approach for relapse prevention via the disruption of the drug-cue memories. We recently demonstrated that retrieval of contextual alcohol memories increased the expression of the mRNA encoding for FK506 binding protein 51 (FKBP51), a regulator of the hypothalamic-pituitary-adrenal (HPA) axis. Here, we explored the role of the HPA axis, and FKBP5/FKBP51 in particular, in the reconsolidation of alcohol memories. We found that the FKBP51 inhibitor SAFit2 given before alcohol-memory retrieval using contextual cues prevented the extinction of alcohol place preference behavior in female mice, suggesting that this protein may play a role in cognitive flexibility in a sex-dependent manner. Conversely, the retrieval of alcohol memories using an odor-taste cue did not affect Fkbp5 expression in rats with a history of chronic alcohol consumption, suggesting that FKBP5 may play a differential role in different alcohol-associated memories. In addition, we provide evidence for HPA axis activation following alcohol memory retrieval, by showing that exposure to an alcohol-associated context led to elevated corticosterone secretion. However, we found that the reconsolidation process was unaffected by HPA axis-related manipulations, namely stress exposure, and administration of corticosterone or the glucocorticoid receptors inhibitor, mifepristone. Our results suggest that although FKBP5 can affect cognitive flexibility, and thereby impact the reconsolidation of alcohol memories, this effect is not likely mediated by HPA axis-related mechanisms.

ERK1/2 inhibition disrupts alcohol memory reconsolidation and prevents relapse
Relapse to alcohol abuse after periods of abstinence, often caused by cue-induced alcohol craving, is a major challenge in the treatment of alcohol addiction. Therefore, disruption of the cue-alcohol associative memories can diminish the risk of relapse. Upon retrieval, memories become temporarily labile before they reconsolidate in a process that requires protein synthesis. Accumulating evidence suggests that the mammalian target of rapamycin complex 1 (mTORC1), which is responsible for the translation of a subset of dendritic proteins, is crucial for memory reconsolidation. Here, we explored the involvement of two regulatory pathways of mTORC1, namely phosphoinositide 3-kinase (PI3K)-AKT and extracellular regulated kinase1/2 (ERK1/2), in the reconsolidation process in a rat model of non-operant alcohol self-administration. We found that retrieval of alcohol memories using an odor-taste cue increased ERK1/2 activation in the amygdala, but did not affect the PI3K-AKT pathway. Importantly, inhibition of ERK1/2 shortly after alcohol memory retrieval impaired reconsolidation and led to long-lasting suppression of relapse to alcohol drinking. Additionally, we show that attenuation of alcohol memories and relapse was also induced by post-retrieval administration of lacosamide, an inhibitor of collapsin response mediator protein-2 (CRMP2) - a translational product of mTORC1 that is functionally regulated by PI3K-AKT signaling. Together, our findings provide evidence for the crucial role of ERK1/2 and CRMP2 in the reconsolidation of alcohol memories, and mark the FDA-approved drug, lacosamide, as a potential treatment for alcohol use disorder.

Distinct effects of priming the brain using tDCS and observational practice: new evidence from brain effective connectivity.
Complex motor skills can be acquired while observing a model without physical practice. Transcranial direct-current stimulation (tDCS) applied to the primary motor cortex (M1) also facilitates motor learning. However, the effectiveness of observational practice for bimanual coordination skills is debated and there is little research on the effects of tDCS on acquiring bimanual skills and the underlying effective/causal brain connectivity. We compared the effect of primary motor cortex tDCS (M1-tDCS) to action-observation (AO) when acquiring a bimanual, two-ball juggling skill and characterized the brain causal connectivity patterns underlying each condition. Twenty healthy young adults with no juggling experience were randomly assigned to either video observation of a skilled juggler or anodal M1-tDCS. Thirty trials of juggling were performed and scored after the intervention. Resting-state EEG data were collected before and after the intervention. Information flow rate was applied to EEG source data to measure causal connectivity. Juggling scores were significantly higher in the AO group (p =.03). We found the strongest information exchange from (L) parietal to (R) parietal regions, strong bidirectional information exchange between (R) parietal and (R) occipital regions and an extensive network of activity that was (L) lateralized in the AO condition. In contrast, the M1-tDCS condition was characterized by bilateral long-range connections with the strongest information exchange from the (R) occipital region to the (R) temporal and (L) occipital regions. This study provides new results about the distinct network dynamics of priming the brain for skill acquisition using direct stimulation or indirect stimulation via action observation.

A generative model of the hippocampal formation trained with theta driven local learning rules
Advances in generative models have recently revolutionised machine learning. Meanwhile, in neuroscience, generative models have long been thought fundamental to animal intelligence. Understanding the biological mechanisms that support these processes promises to shed light on the relationship between biological and artificial intelligence. In animals, the hippocampal formation is thought to learn and use a generative model to support its role in spatial and non-spatial memory. Here we introduce a biologically plausible model of the hippocampal formation tantamount to a Helmholtz machine that we apply to a temporal stream of inputs. A novel component of our model is that fast theta-band oscillations (5-10 Hz) gate the direction of information flow throughout the network, training it akin to a high-frequency wake-sleep algorithm. Our model accurately infers the latent state of high-dimensional sensory environments and generates realistic sensory predictions. Furthermore, it can learn to path integrate by developing a ring attractor connectivity structure matching previous theoretical proposals and flexibly transfer this structure between environments. Whereas many models trade-off biological plausibility with generality, our model captures a variety of hippocampal cognitive functions under one biologically plausible local learning rule.

Lumbosacral spinal cord functional connectivity at rest: From feasibility to reliability
In the past decade, exploration of spontaneous blood-oxygen-level-dependent (BOLD) signal fluctuations has expanded beyond the brain to include the spinal cord. While most studies have predominantly focused on the cervical region, the lumbosacral segments play a crucial role in motor control and sensory processing of the lower limbs. Addressing this gap, the aims of the current study were two-fold: first, confirming the presence and nature of organized spontaneous BOLD signals in the human lumbosacral spinal cord; second, systematically assessing the impact of various denoising strategies on signal quality and functional connectivity (FC) patterns. Given the susceptibility of spinal cord fMRI to noise, this step is pivotal to ensure the robustness of intrinsic FC. Our findings uncovered bilateral FC between the ventral horns. Importantly, these patterns were consistently observed across denoising methods and demonstrating fair to excellent reliability. Conversely, no other significant connectivity patterns were identified across the remaining horns. Importantly, the evaluation of diverse denoising strategies highlighted the efficacy of PNM-based pipelines in cleaning the signal while preserving the strength and reliability of connectivity estimates. Together, our results provide evidence of robust FC patterns in the lumbosacral spinal cord, thereby paving the way for future studies probing caudal spinal activity.

Differential developmental blueprints of organ-intrinsic nervous systems
The organ-intrinsic nervous system is a major interface between visceral organs and the brain, mediating important sensory and regulatory functions in the body-brain axis and serving as critical local processors for organ homeostasis. Molecularly, anatomically, and functionally, organ-intrinsic neurons are highly specialized for their host organs. However, the underlying mechanism that drives this specialization is largely unknown. Here, we describe the differential strategies utilized to achieve organ-specific organization between the enteric nervous system (ENS)1 and the intrinsic cardiac nervous system (ICNS)2, a neuronal network essential for heart performance but poorly characterized. Integrating high-resolution whole-embryo imaging, single-cell genomics, spatial transcriptomics, proteomics, and bioinformatics, we uncover that unlike the ENS which is highly mobile and colonizes the entire gastrointestinal (GI) tract, the ICNS uses a rich set of extracellular matrix (ECM) genes that match with surrounding heart cells and an intermediate dedicated neuronal progenitor state to stabilize itself for a "beads-on-the-necklace" organization on heart atria. While ICNS- and ENS-precursors are genetically similar, their differentiation paths are influenced by their host-organs, leading to distinct mature neuron types. Co-culturing ENS-precursors with heart cells shifts their identity towards the ICNS and induces the expression of heart-matching ECM genes. Our cross-organ study thus reveals fundamental principles for the maturation and specialization of organ-intrinsic neurons.

Distributed Representations for Cognitive Control in Frontal Medial Cortex
In natural and artificial neural networks, modularity and distributed structure afford complementary but competing benefits. The former allows for hierarchical representations that can flexibly recombine modules to address novel problems, whereas the latter affords better generalization. Here we investigate these competing demands in the context of sequential behavior. First, we explore this by comparing the properties of several recurrent neural network models. We find that explicit hierarchical structure fails to provide an advantage for generalization above a 'flat' model that does not incorporate hierarchical structure. However, hierarchy appears to facilitate cognitive control processes that support non-routine behaviors and behaviors that are carried out under computational stress. Second, we compare these models against functional magnetic resonance imaging (fMRI) data using representational similarity analysis. We find that a model that incorporates so-called wiring costs in the cost function, which produces a hierarchically-organized gradient of representational structure across the hidden layer of the neural network, best accounts for fMRI data collected from human participants in a previous study (Holroyd et al., 2018). The results reveal that the anterior cingulate cortex (ACC) encodes distributed representations of sequential task context along a rostro-caudal gradient of abstraction: rostral ACC encodes relatively abstract and temporally extended patterns of activity compared to those encoded by caudal ACC. These results provide insight into the role of ACC in motivation and cognitive control.

The association of longitudinal diet and waist-to-hip ratio from midlife to old age with hippocampus connectivity and memory in old age: a cohort study
Background: Epidemiological studies suggest lifestyle factors may reduce the risk of dementia. However, few studies have examined the association of diet and waist-to-hip ratio with hippocampus connectivity. Methods: In the Whitehall II Imaging Sub-study, we examined longitudinal changes in diet quality in 512 participants and waist-to-hip ratio in 665 participants. Diet quality was measured using the Alternative Health Eating Index-2010 assessed three times across 11 years between ages 48 and 60 years, and waist-to-hip ratio five times over 21 years between ages 48 and 68 years. Brain imaging and cognitive tests were performed at age 70{+/-}5 years. We measured white matter using diffusion tensor imaging and hippocampal functional connectivity using resting-state functional magnetic resonance imaging. In addition to associations of diet and waist-to-hip ratio with brain imaging measures, we tested whether associations between diet, waist-to-hip ratio and cognitive performance were mediated by brain connectivity. Findings: Better diet quality in midlife and improvements in diet over mid-to-late life were associated with higher hippocampal functional connectivity to the occipital lobe and cerebellum, and better white matter integrity as measured by higher fractional anisotropy and lower diffusivity. Higher waist-to-hip ratio in midlife was associated with higher mean and radial diffusivity and lower fractional anisotropy in several tracts including the inferior longitudinal fasciculus and cingulum. Associations between midlife waist-to-hip ratio, working memory and executive function were partially mediated by radial diffusivity. Interpretation: Healthier diets and waist-to-hip ratios throughout midlife are associated with better brain health later in life.