bioRxiv Subject Collection: Neuroscience's Journal

Low dose of a non-urea selective GIRK channel activator improves hippocampal-dependent synaptic plasticity and memory disrupted by amyloid-β oligomers
Increased neural activity characterizes early Alzheimers disease (AD), serving as a prognostic indicator for disease progression and cognitive decline. Mechanisms that drive this hyperactivity and their behavioral effects remain mostly unrevealed, although normalizing altered excitability levels has been shown to reverse cognitive impairment in early AD, both in animals and humans. Soluble amyloid-{beta} oligomers (oA{beta}) primary accumulate in limbic regions like hippocampus and induce neuronal hyperexcitability and subsequent cognitive deficits by impairing ion channel s function. Indeed, G protein-gated inwardly rectifying K+ (GIRK) channels -that control neuronal excitability- are greatly affected and their selective pharmacological activation has already been shown very effective to counteract oA{beta}-induced hyperexcitability and hippocampal dysfunction. However, GIRK gain-of-function in healthy animals disrupts learning, memory and underlying synaptic plasticity, greatly limiting its therapeutic potential in preclinical asymptomatic early AD patients. Therefore, GIRK-based pharmacological treatment needs further investigation to overcome these limitations. Here we tested two doses of a novel, more potent, and neuronal selective GIRK activator, VU0810464, in healthy and early oAB-generated AD male and female mice. Both doses normalized hippocampal synaptic plasticity (long-term potentiation, LTP) and associated spatial object location memory (OLM) without sex dimorphism in AD animals. However, in healthy mice, low VU0810464 dose did not significantly alter LTP and OLM, whereas the high dose disrupted both. Our results suggest that the precise tuning of neural excitability with low dosing of VU0810464 might be a promising strategy to safely treat and prevent hippocampal overexcitation and upstreaming memory deficits in early preclinical asymptomatic phases of AD.

A neural network model that generates salt concentration memory-dependent chemotaxis in Caenorhabditis elegans
A neuroanatomical minimal network model was revisited to elucidate the mechanism of salt concentration memory-dependent chemotaxis observed in Caenorhabditis elegans. C. elegans memorizes the salt concentration during cultivation, manifesting a pronounced taste preference for this concentration. The right-side head sensory neuron, designated ASER, exhibits a response to a decrease in salt concentration. The basal level of glutamate transmission from ASER has been demonstrated to transiently increase and decrease when the current environmental salt concentrations are lower and higher, respectively, than that during previous cultivation. Given the sensitivity of excitatory/inhibitory glutamate receptors expressed on the postsynaptic AIY interneurons, it can be anticipated that the ASER-AIY synaptic transmission will undergo a reversal due to alterations in the basal glutamate release. The neural network model, derived with the hypothesis, reproduced the salt concentration memory-dependent preference behavior and revealed the modular neural circuit function downstream of ASE that is responsible for salt klinotaxis.

Neural coding of choice and outcome are modulated by uncertainty in orbitofrontal but not secondary motor cortex
Orbitofrontal cortex (OFC) and secondary motor cortex (M2) are both implicated in flexible reward learning but the conditions that differentially recruit these regions are not fully understood. We imaged calcium activity from single neurons in OFC or M2 during de novo learning of uncertain reward probability schedules. After controlling for experience, predictions of choice were decoded from M2 neurons with similar accuracy under all certainty conditions, but were more accurately decoded from OFC neurons under greater uncertainty. In M2, the proportion of outcome selective neurons decreased with uncertainty whereas this proportion remained stable in OFC, due to an increased recruitment of reward-selective neurons across levels of uncertainty. Decoding accuracy of both choice and outcome were positively correlated with indices of flexible strategy use in OFC, but not M2. Our results indicate that OFC neurons preferentially encode choices and outcomes under conditions of uncertainty that foster greater reliance on adaptive strategies.

Microglia modulate cerebral blood flow and neurovascular coupling through ectonucleotidase CD39
Microglia and the border-associated macrophages (BAMs) contribute to the modulation of cerebral blood flow (CBF), but the mechanisms have remained ill-defined. Here, we show that microglia regulate the CBF baseline and upsurges after whisker stimulation or intracisternal magna injection of adenosine triphosphate (ATP). Genetic or pharmacological depletion of microglia reduces the activity-dependent hyperemia but not the cerebrovascular responses to adenosine stimulation. Notably, microglia repopulation corrects these CBF reactivity deficits. The microglial-dependent regulation of CBF requires the ATP-sensing P2ry12 receptor and the ectonucleotidase CD39 that initiates the breakdown of extracellular ATP. Pharmacological inhibition or microglia-specific deletion of CD39 simulates the CBF anomalies detected in microglia-deficient mice and reduces the rise of extracellular adenosine after whisker stimulation. Together, these results suggest that the microglial CD39-initiated conversion of extracellular ATP to adenosine is an important step in neurovascular coupling and the regulation of cerebrovascular reactivity.

Effects of HA1 (a Probucol Analogue) and ApoC3 siRNA on Lipoprotein-Amyloid Metabolism, Neurovascular Integrity and Cognitive Function in db/db Mice
Abstract Background and aims Chronically elevated levels of circulating lipoprotein-amyloid-beta (Abeta) are implicated in the disruption of the blood-brain barrier and the initiation of a neurodegenerative cascade leading to Alzheimers disease (AD). Type 2 diabetes is associated with BBB dysfunction, dyslipidaemia, and an increased risk of AD. However, alterations in triglyceride-rich lipoproteins (TRL)-Abeta homeostasis and its downstream effects on the BBB in a diabetes context remain explored. This study aimed to 1) investigate, in a preclinical model of diabetes, the hypothesis that diabetes-induced impairments in TRL-Abeta metabolism might compromise BBB integrity and exacerbate cognitive function and behavioural changes, and 2) assess the efficacy of interventions that improve TRL catabolism, including probucol, HA1, and ApoC3 siRNA, to prevent disease progression by lowering circulating TRL-Abeta levels. Methods Five-week-old db/db mice underwent a 9- or 23-weeks dietary interventions with probucol, a probucol prodrug HA1, or a standard diet with four-weekly injections of ApoC3 siRNA. Db/+ mice served as negative controls for each treatment duration. Blood levels of Abeta and ApoB were measured using ELISA. Immunofluorescence imaging was used to quantify enterocytic levels of Abeta and ApoB, and assess changes in neurovascular integrity (IgG, PDGFRbeta, ZO1, occludin), neuroinflammation (GFAP, Iba1), and cerebral oxidative stress (8OHdG). Results Our results indicate that diabetes increased the abundance of plasma amyloid, specifically Abeta42, which correlated with enterocytic abundance, suggesting exaggerated postprandial excretion. Disrupted plasma amyloid homeostasis was associated with BBB breakdown, including diminished barrier function, and the loss of pericytes and astrocytes. Provision of the probucol analogue, HA1, normalised plasma and enterocytic amyloidemia concomitant with the preservation of the neurovascular junction. Treatment with ApoC3 siRNA attenuated plasma Abeta42 and modestly reduced neurovascular inflammation. Conclusion The findings further support the hypothesis that aberrant peripheral metabolism of lipoprotein-Abeta is associated with microvascular corruption and the development of anxiety-like behaviour. HA1 is more effective than probucol or ApoC3 siRNA in positively modulating lipoprotein-amyloid homeostasis in db/db mice and maintaining central capillary integrity.

Molecular impact of nicotine and smoking exposure on the developing and adult mouse brain
Maternal smoking during pregnancy (MSDP) is associated with significant cognitive and behavioral effects on offspring. While neurodevelopmental outcomes have been studied for prenatal exposure to nicotine, the main psychoactive component of cigarette smoke, its contribution to MSDP effects has never been explored. Comparing the effects of these substances on molecular signaling in the prenatal and adult brain may provide insights into nicotinic and broader tobacco consequences that are developmental-stage specific or age-independent. Pregnant mice were administered nicotine or exposed to chronic cigarette smoke, and RNA-sequencing was performed on frontal cortices of postnatal day 0 pups born to these mice, as well as on frontal cortices and blood of the adult dams. We identified 1,010 and 4,165 differentially expressed genes (DEGs) in nicotine and smoking-exposed pup brains, respectively (FDR<0.05, Ns = 19 nicotine-exposed vs 23 vehicle-exposed; 46 smoking-exposed vs 49 controls). Prenatal nicotine exposure (PNE) alone was related to dopaminergic synapses and long-term synaptic depression, whereas MSDP was associated with the SNARE complex and vesicle transport. Both substances affected SMN-Sm protein complexes and postsynaptic endosomes. Analyses at the transcript, exon, and exon-exon junction levels supported gene level results and revealed additional smoking-affected processes. No DEGs at FDR<0.05 were found in adult mouse brain for any substance (12 nicotine-administered vs 11 vehicle-administered; 12 smoking-exposed vs 12 controls), nor in adult blood (12 smoking-exposed vs 12 controls), and only 3% and 6.41% of the DEGs in smoking-exposed pup brain replicated in smoking-exposed blood and human prenatal brain, respectively. Together, these results demonstrate variable but overlapping molecular effects of PNE and MSDP on the developing brain, and attenuated effects of both smoking and nicotine on adult versus fetal brain.

Flexible 3D kirigami probes for in vitro and in vivo neural applications
Three-dimensional (3D) microelectrode arrays (MEAs) are gaining popularity as brain-machine interfaces and platforms for studying electrophysiological activity. Interactions with neural tissue depend on the electrochemical, mechanical, and spatial features of the recording platform. While planar or protruding two-dimensional MEAs are limited in their ability to capture neural activity across layers, existing 3D platforms still require advancements in manufacturing scalability, spatial resolution, and tissue integration. In this work, we present a customizable, scalable, and straightforward approach to fabricate flexible 3D kirigami MEAs containing both surface and penetrating electrodes, designed to interact with the 3D space of neural tissue. These novel probes feature up to 512 electrodes distributed across 128 shanks in a single flexible device, with shank heights reaching up to 1 mm. We successfully deployed our 3D kirigami MEAs in several neural applications, both in vitro and in vivo, and identified spatially dependent electrophysiological activity patterns. Flexible 3D kirigami MEAs are therefore a powerful tool for large-scale electrical sampling of complex neural tissues while improving tissue integration and offering enhanced capabilities for analyzing neural disorders and disease models where high spatial resolution is required.

Oral Boldine Administration Attenuates Glia Activation and Prevents Neuropathic Pain in a Murine Spared Nerve Injury Model
Chronic pain is present in about 20% of the population and is a major burden to the health care system. About 30-40% of these patients report neuropathic pain. Neuropathic pain is defined as pain caused by injury or disease of the somatosensory nervous system. Current available treatments for neuropathic pain have limited efficacy and substantial side effects. To address the need for more effective and safer treatments for neuropathic pain, this study aimed to test whether boldine, a naturally occurring alkaloid, could attenuate neuropathic pain in a murine model of spared nerve injury (SNI). We found that oral administration of boldine at 50 mg/kg body weight/day resulted in significant reduction of SNI-induced mechanical and thermal hypersensitivity. Boldine also corrected SNI-induced weight bearing deficits, which are an indication of spontaneous pain. Boldine significantly inhibited SNI-induced peripheral inflammation as indicated by reduced levels of inflammatory cytokines/chemokines in the serum. Immunofluorescence studies revealed that boldine reduced the number of reactive astrocytes and inhibited microglia activation in the dorsal horn of lumbar spinal cord. Boldine also reduced the mRNA level of pro-inflammatory markers including IL-1{beta} and TNF- in the lumbar spinal cord after SNI. Quantitative PCR showed that boldine inhibited the lipopolysaccharide-induced overexpression of inflammatory markers in primary mouse astrocytes and in BV-2 microglial cells. Our findings suggest that boldine may be a promising therapeutic candidate for the treatment of neuropathic pain, possibly through inhibition of glia activation and neuroinflammation.

Mitochondrial dysfunction Impairs the Nuclear Pore Complex in Parkinson's Disease Pathogenesis
The mislocalization of transcription factors and irregularities in the nuclear envelope of dopaminergic neurons affected by Parkinson's disease (PD) implicates the nuclear pore in disease pathogenesis. While mitochondrial dysfunction is an integral component of PD pathophysiology, the involvement of channel-forming nucleoporins (Nups) in mitochondrial dysfunction-related neurodegeneration has not been investigated. Here we have identified pathological changes in the levels and distribution of a set of Nups, which are key structural and functional components of the nuclear pore complex, in dopaminergic neuronal models of PD. We observed that mitochondrial dysfunction reduces the expression of these Nups and disrupts the localization of Ran GTPase in both in vitro and in vivo dopaminergic neuron models. Furthermore, the nuclear pore central channel component, Nup62, mislocalizes and accumulates in the cytoplasm of these neurons under mitochondrial stress conditions. Mitochondrial stress also interferes with classical nuclear export of proteins in dopaminergic neural cells. Notably, we observed Nup pathology and Ran gradient loss in nigral dopaminergic neurons of PD patient brains, which highlights the clinical relevance of nuclear pore dysfunction as a disease mechanism. These findings provide direct evidence of the critical role of Nup-related abnormalities in mitochondrial dysfunction-induced degeneration of dopaminergic neurons, ultimately connecting nuclear pore complex and nucleocytoplasmic transport dysregulation to cell death in the development of PD.

Selective Enhancement of the Interneuron Network and Gamma-Band Power via GluN2C/GluN2D NMDA Receptor Potentiation
N-methyl-D-aspartate receptors (NMDARs) comprise a family of ligand-gated ionotropic glutamate receptors that mediate a slow, calcium-permeable component to excitatory neurotransmission. The GluN2D subunit is enriched in GABAergic inhibitory interneurons in cortical tissue. Diminished levels of GABAergic inhibition contribute to multiple neuropsychiatric conditions, suggesting that enhancing inhibition may have therapeutic utility, thus making GluN2D modulation an attractive drug target. Here, we describe the actions of a GluN2C/GluN2D-selective positive allosteric modulator (PAM), (+)-EU1180-453, which has improved drug-like properties such as increased aqueous solubility compared to the first-in-class GluN2C/GluN2D-selective prototypical PAM (+)-CIQ. (+)-EU1180-453 doubles the NMDAR response at lower concentrations (< 10 uM) compared to (+)-CIQ, and produces a greater degree of maximal potentiation at 30 uM. Using in vitro electrophysiological recordings, we show that (+)-EU1180-453 potentiates triheteromeric NMDARs containing at least one GluN2C or GluN2D subunit, and is active at both exon5-lacking and exon5-containing GluN1 splice variants. (+)-EU1180-453 increases glutamate efficacy for GluN2C/GluN2D-containing NMDARs by both prolonging the deactivation time and potentiating the peak response amplitude. We show that (+)-EU1180-453 selectively increases synaptic NMDAR-mediated charge transfer onto P11-15 CA1 stratum radiatum hippocampal interneurons, but is without effect on CA1 pyramidal cells. This increased charge transfer enhances inhibitory output from GABAergic interneurons onto CA1 pyramidal cells in a GluN2D-dependent manner. (+)-EU1180-453 also shifts excitatory-to-inhibitory coupling towards increased inhibition and produces enhanced gamma band power from carbachol-induced field potential oscillations in hippocampal slices. Thus, (+)-EU1180-453 can enhance overall circuit inhibition, which could prove therapeutically useful for the treatment of anxiety, depression, schizophrenia, and other neuropsychiatric disorders.

DNA replication fork speed Acts as an Engine in Cortical Neurogenesis
DNA fork speed, the rate of replication fork progression, has emerged as a cellular plasticity regulator, however, for its role in neurogenesis has never been explored before. Here, we show that fork speed was increased as neural progenitors-radial glial cells (RGCs) transition from symmetric to asymmetric divisions. After selectively deleting mini-chromosome maintenance complex (MCMs)-binding protein (MCMBP), fork speed was increased in RGCs, resulted in widespread apoptosis, DNA damage and micronuclei at later stage of neurogenesis, which triggered p53 activation and led to microcephaly. Further, co-deletion of Trp53 with Mcmbp largely rescued brain phenotype, however, fork speed became faster, unexpectedly resulting in massive RGCs detachment from their resident place. Mechanistically, MCM3 can interact with p53 mediating centrosome biogenesis to anchor RGCs during DNA replication. Finally, behavior analysis indicated that fast fork speed led to an anxiety-like behavior in mice. Altogether, our results illuminate an unrecognized role about DNA fork speed in corticogenesis.

A cross-species analysis of neuroanatomical covariance sex difference in humans and mice
Structural covariance in brain anatomy is thought to reflect inter-regional sharing of developmental influences - although this hypothesis has proved hard to causally test. Here, we use neuroimaging in humans and mice to study sex-differences in anatomical covariance - asking if regions that have developed shared sex differences in volume across species also show shared sex difference in volume covariance. The study design illuminates both the biology of sex-differences and theoretical models for anatomical covariance -- benefitting from tests of inter-species convergence. We find that volumetric structural covariance is stronger in adult females compared to adult males for both wild-type mice and healthy human subjects: 98% of all comparisons with statistically significant covariance sex differences in mice are female-biased, while 76% of all such comparisons are female-biased in humans (q < 0.05). In both species, a region's covariance and volumetric sex-biases have weak inverse relationships to each other: volumetrically male-biased regions contain more female-biased covariation, while volumetrically female-biased regions have more male-biased covariation (mice: r = -0.185, p = 0.002; humans: r = -0.189, p = 0.001). Our results identify a conserved tendency for females to show stronger neuroanatomical covariance than males, evident across species, which suggests that stronger structural covariance in females could be an evolutionarily conserved feature that is partially related to volumetric alterations through sex.

A spatial single-cell atlas of the claustro-insular region uncovers key regulators of neuronal identity and excitability
The claustro-insular region is an evolutionarily conserved and extensively interconnected brain area, critical for functions such as attention, cognitive flexibility, interoception, and affective processing. Despite its importance, its cellular composition and organization remain poorly characterized, hindering a comprehensive understanding of the mechanisms underlying its diverse functions. By combining single-cell RNA sequencing and spatial transcriptomics, we created a high-resolution atlas of this region in mice, uncovering distinct neuronal subtypes and unexpected complexity. Leveraging this atlas, we investigated the role of NR4A2, a neuropsychiatric risk factor expressed in several claustro-insular neuronal subtypes. In an Nr4a2 haploinsufficiency model, we found that only claustrum neurons exhibited shifts in molecular identity. This identity shift, which involved the activation of a transcription factor cascade, was associated with alterations in neuronal firing activity. Our findings provide new insights into the cellular architecture of the claustro-insular region and highlights Nr4a2 as a master regulator of its component's identities.

Inhibition of β-catenin signaling by Amyloid-β in endothelial cells impairs vascular barrier integrity
Dysfunction of the blood-brain barrier (BBB) is emerging as a critical mediator of Alzheimer's Disease (AD) progression and precedes the formation of large Amyloid-{beta} (A{beta}) plaques in AD patients. Maintenance of the blood brain barrier integrity by brain microvascular endothelial cells (BMECs) is essential for brain homeostasis and is in part maintained by WNT/{beta}-catenin signaling. We hypothesized that A{beta} induces blood-brain barrier dysfunction by inhibiting WNT/{beta}-catenin signaling in brain endothelial cells, leading to reduced expression of BBB-related genes and barrier function. In brain endothelial cells, we found that A{beta} directly decreased {beta}-catenin transcriptional activity, but also inhibited activation of {beta}-catenin by the WNT3A peptide. Additionally, A{beta} reduced barrier integrity of ECs, which was in part restored by exposure to exogenous WNT3A. We observed A{beta}-induced inhibition within 3 hours of exposure. A{beta}-induced inhibition of {beta}-catenin signaling was consistent across both immortalized brain ECs and ECs differentiated from human induced pluripotent stem cells(iPSCs). We next investigated human iPSCs with and without the N141I mutation in presenilin-2 (PSEN2), known to increase A{beta} buildup and familial AD risk. We found the AD ECs showed smaller cell size and expressed lower VE-cadherin at the protein level. RNA sequencing revealed that the presence of PSEN2 N141I did not significantly impact gene expression of genes coding for proteins canonically involved in barrier integrity but did show differences in the expression of transposable elements. These results indicate that BBB dysfunction early in AD may be the result of A{beta} in the brain impairing WNT/{beta}-catenin signaling which maintains the barrier integrity of brain endothelial cells.

Presaccadic attentional shifts are not modulated by saccade amplitude
Humans constantly explore the visual environment through saccades, bringing relevant visual stimuli to the center of the gaze. Before the eyes begin to move, visual attention is directed to the intended saccade target. As a consequence of this presaccadic shift of attention (PSA), visual perception is enhanced at the future gaze position. PSA has been investigated in a variety of saccade amplitudes, from microsaccades to locations that exceed the oculomotor range. Interestingly, recent studies have shown that PSA effects on visual perception are not equally distributed around the visual field. However, it remains unknown whether the magnitude of presaccadic perceptual enhancement varies with the amplitude of the saccades. Here, we measured contrast sensitivity thresholds during saccade planning in a two-alternative forced-choice (2AFC) discrimination task in human observers. Filtered pink noise (1/f) patches, presented at four eccentricities scaled in size according to the cortical magnification factor were used as visual targets. This method was adopted to mitigate well-known eccentricity effects on perception, thereby enabling us to explore the effects associated to saccade amplitudes. First, our results show that saccade preparation enhanced contrast sensitivity in all tested locations. Importantly, we found that this presaccadic perceptual enhancement was not modulated by the amplitude of the saccades. These findings suggest that presaccadic attention operates consistently across different saccade amplitudes, enhancing visual processing at intended gaze positions regardless of saccade size.

Efficient laminar-distributed interactions and orientation selectivity in the mouse V1 cortical column
The emergence of orientation selectivity in the visual cortex is a well-known phenomenon in neuroscience, but the details of such emergence and the role of different cortical layers and cell types, particularly in rodents which lack a topographical organization of orientation-selectivity (OS) properties, are less clear. To tackle this question, we use an existing biologically detailed model of the mouse V1 cortical column, which is constrained by existing connectivity data across cortical layers and between pyramidal, PV, SST and VIP cell types. Using this model as a basis, we implemented activity-dependent structural plasticity induced by stimulation with orientated drifting gratings, leading to a good match of tuning properties of pyramidal cells with experimentally observed OS laminar distribution, their evoked firing rate and tuning width. We then employed a mean-field model to uncover the role of co-tuned subnetworks in laminar signal propagation and explain the effects of intra- and inter- laminar coupling distributions. Our plasticity-induced modified model and mean-field model were able to explain both the excitatory enhancement through co-tuned subnetworks and inter-laminar disynaptic inhibition. Overall, our work highlights the importance of the clustering of neural selectivity features for effective excitatory transmission in cortical circuits.

Unraveling eye movement-related eardrum oscillations (EMREOs): how saccade direction and middle ear properties shape amplitude and time course
Eye movement-related eardrum oscillations (EMREOs) reflect movements of the tympanic membrane that are known to scale with the magnitude and direction of saccades. While EMREOs have been consistently described in humans and non-human primates, many questions regarding this phenomenon remain open. Based on bilateral in-ear recordings in human participants we here probe a number of their properties that improve our understanding of the EMREOs' origin and functional significance. We show that the time courses are comparable between the left and right ears, and between paradigms guiding saccades by visual and auditory targets. However, the precise time course and amplitude differ significantly between ipsi- and contralateral saccades beyond the previously known phase-inversion described for saccades in opposing directions. Finally, we show that the EMREO amplitude is negatively related to the compliance of the tympanic membrane. These results altogether raise potential challenges for the interpretation of EMREOs in the light of a coordinate-alignment between vision and hearing. Furthermore, they support the notion that EMREOs largely reflect motor-related top-down signals that are relayed to the middle ear muscles in a differential principle similar as governing the execution of ipsi- and contralateral saccades.

In vivo cell-type and brain region classification via multimodal contrastive learning
Current electrophysiological approaches can track the activity of many neurons, yet it is usually unknown which cell-types or brain areas are being recorded without further molecular or histological analysis. Developing accurate and scalable algorithms for identifying the cell-type and brain region of recorded neurons is thus crucial for improving our understanding of neural computation. In this work, we develop a multimodal contrastive learning approach for neural data that can be fine-tuned for different downstream tasks, including inference of cell-type and brain location. We utilize this approach to jointly embed the activity autocorrelations and extracellular waveforms of individual neurons. We demonstrate that our embedding approach, Neuronal Embeddings via MultimOdal contrastive learning (NEMO), paired with supervised fine-tuning, achieves state-of-the-art cell-type classification for an opto-tagged visual cortex dataset and brain region classification for the public International Brain Laboratory brain-wide map dataset. Our method represents a promising step towards accurate cell-type and brain region classification from electrophysiological recordings.

Sufficiency of ITD for (biased) human auditory azimuthal perception
Experiments on human auditory perception have shown that interaural time difference (ITD) is sufficient to generate spatial percepts, even though stimuli containing only the ITD cue are perceived as being emitted from inside the head instead of from external locations at specific azimuths. These experiments are thus interpreted as "lateralization" instead of "localization" tasks. In fact, lateralized spatial perception has been quantified using tasks in which participants have to report their estimates by selecting a putative location inside the head, or matching the perceived position to sounds with a given interaural level difference. Therefore, these estimates are made with respect to internal frames of reference, but it is unclear whether these percepts have any significance for the more ecological problem of locating an external sound source. In order to investigate the link between internalized spatial percepts and sound localization, we designed a new task in which subjects are instructed to report externalized azimuthal location for sounds containing only ITD cues. Despite the mismatch between an internalized percept having to be reported as emanating from an external location, subjects were able to estimate azimuths consistently. Furthermore, normalized estimates were indistinguishable from those obtained using traditional lateralization tasks. Our results revealed a direct relationship between perceived azimuths and ITD, which deviates from that obtained from acoustical analysis of binaural recordings, revealing estimation biases. Intriguingly, these results indicate that externalized percepts are not required for the generation of azimuthal percepts.

Chemogenetic manipulation of the ventral pallidum-nucleus accumbens shell pathway modulates sucrose consumption independent of motivation in female rats.
Here we investigated the role of the ventral pallidum (VP) to nucleus accumbens shell (AcbSh) pathway in mediating sucrose consumption and motivation for food in female rats. Using chemogenetic techniques, excitatory and inhibitory designer receptors were expressed in VP neurons projecting to the AcbSh. Behavioral assays assessed both lever pressing under a progressive ratio schedule and free sucrose consumption following activation or inhibition of this pathway. Results showed that inhibition of VP-AcbSh projections increased sucrose intake, while excitation reduced it, without affecting motivation to work for food. These findings indicate a specific influence of the VP-AcbSh pathway on sucrose consumption independent of food-seeking motivation. The study highlights the importance of differentiating VP efferents in understanding distinct roles in feeding behaviors and suggests that the VP-AcbSh pathway modulates consumption based on food type and potentially other variables.

Contactin-1 is a critical neuronal cell surface receptor for perineuronal net structure
Perineuronal nets (PNNs), are neuron-specific substructures within the neural extracellular matrix (ECM). These reticular structures form on a very small subset of neurons in the central nervous system (CNS) and yet have a profound impact in regulating neuronal development and physiology. PNNs are well-established as key regulators of plasticity in the CNS. Their appearance coincides with the developmental transition of the brain more to less plastic state. And, importantly, numerous studies have demonstrated that indeed PNNs play a primary role in regulating this transition. There is, however, a growing literature implicating PNNs in numerous roles in neural physiology beyond their role in regulating developmental plasticity. Accordingly, numerous studies have shown PNNs are altered in a variety of neurological and neuropsychiatric diseases, linking them to these conditions. Despite the growing interest in PNNs, the mechanisms by which they modulate neural functions are poorly understood. We believe the limited mechanistic understanding of PNNs is derived from the fact that there are limited models, tools or techniques that specifically target PNNs in a cell-autonomous manner and without also disrupting the surrounding neural ECM. These limitations are primarily due to our incomplete understanding of PNN composition and structure. In particular, there is little understanding of the neuronal cell surface receptors that nucleate these structures on subset of neurons on which they form in the CNS. Therefore, the main focus our work is to identify the neuronal cell surface proteins critical for PNN formation and structure. In our previous studies we demonstrated PNN components are immobilized on the neuronal surface by two distinct mechanisms, one dependent on the hyaluronan backbone of PNNs and the other mediated by a complex formed by receptor protein tyrosine phosphatase zeta (RPTP{zeta}) and tenascin-R (Tnr). Here we first demonstrate that the Tnr-RPTP{zeta} complex in PNNs is bound to the cell surface by a glycosylphosphatidylinositol (GPI)-linked receptor protein. Using a biochemical and structural approach we demonstrate the GPI-linked protein critical for binding the Tnr-RPTP{zeta} complex in PNNs is contactin-1 (Cntn1). We further show the binding of this complex in PNNs by Cntn1 is critical for PNN structure. We believe identification of CNTN1 as a key cell-surface protein for PNN structure is a very significant step forward in our understanding of PNN formation and structure and will offer new strategies and targets to manipulate PNNs and better understand their function.

Restoring and sustaining human postmortem retinal light responses with scalable methods for testing degenerative disease therapies
Neuro- and retinal degenerative diseases rob millions of aging individuals of their independence. Researching these diseases in human tissue has been hindered by the immediate loss of electric activity in neurons after the circulation ceases. Recent studies indicate that limited neuronal activity can be revived postmortem, even in the retina. We capitalized on this discovery by successfully restoring and maintaining in vivo-like light responses in eyes recovered up to four hours and stored for up to 48 hours postmortem. This breakthrough significantly increases the availability of functionally viable human retinas for research. Our AI-based postmortem retinal imaging platform identifies retinal structures and allows us to compare light responses in the healthy central and peripheral retina with ex vivo electroretinography. We use this platform to measure the dark adaptation of human macular cones from controls and donors with age-related macular degeneration for the first time in the absence of the retinal pigment epithelium. We developed increased throughput technology and a model to simulate disease-associated acute ischemia-reperfusion. In this model, we demonstrate the protective or toxic effects of several drugs targeting oxidative stress or glutamate excitotoxicity.

Neural markers of abstinence in alcohol dependence: Insights from Reward Learning
Maladaptive reward learning and decision-making circuity are key factors in the onset and progression of alcohol use disorder and have therefore emerged as key targets for neuropsychological and pharmacological interventions. Probabilistic reversal learning studies have consistently reported impaired learning in recently detoxified alcohol dependent (AD) participants. However, the neural and behavioural changes associated with reward learning which occur throughout abstinence remain unexplored. Here, we show that AD participants, with mean abstinence of 20 months, exhibit intact behavioural performance within an electroencephalography (EEG) probabilistic reversal learning task. Reinforcement learning modelling reveals reward and punishment related learning rates and exploration rates are comparable between AD and healthy control (HC) participants, suggesting recovery of even the nuanced aspects of learning in longer term abstinence. However, EEG analysis indicates that AD, compared to HC participants, show globally elevated event-related potential (ERP) feedback related negativity (FRN) following reward valuation. Furthermore, Feedback-P3 valence prediction error signal is negatively associated with abstinence duration indicating a potential state marker of AD recovery. We then employ unsupervised machine learning (canonical polyadic tensor decomposition) to identify spatiotemporal EEG patterns of reward valuation in a purely data-driven manner. Classification analysis shows these tensor components can predict group membership with 80.4% accuracy. By probing group differences in tensor components, we discover early hyperfunctioning in centro-frontal regions linked to alcohol dependence and associated with early abstinence. The clinically meaningful EEG biomarkers presented here could guide the development of more targeted treatments and support big data approaches to objective patient monitoring.

Real-Time Closed-Loop Feedback System For Mouse Mesoscale Cortical Signal And Movement Control: CLoPy
We present the implementation and efficacy of an open-source closed-loop neurofeedback (CLNF) and closed-loop movement feedback (CLMF) system. In CLNF, we measure mm-scale cortical mesoscale activity with GCaMP6s and provide graded auditory feedback (within ~50 ms) based on changes in dorsal-cortical activation within regions of interest (ROI) and with a specified rule. Single or dual ROIs (ROI1, ROI2) on the dorsal cortical map were selected as targets. Both motor and sensory regions supported closed-loop training in male and female mice. Mice modulated activity in rule-specific target cortical ROIs to get increasing rewards over days (RM ANOVA p=2.83e-5) and adapted to changes in ROI rules (RM ANOVA p=8.3e-10, Table 4 for different rule changes). In CLMF, feedback was based on tracking a specified body movement, and rewards were generated when the behavior reached a threshold. For movement training, the group that received graded auditory feedback performed significantly better (RM-ANOVA p=9.6e-7) than a control group (RM-ANOVA p=0.49) within four training days. Additionally, mice can learn a change in task rule from left forelimb to right forelimb within a day, after a brief performance drop on day 5. Offline analysis of neural data and behavioral tracking revealed changes in the overall distribution of dF/F0 values in CLNF and body-part speed values in CLMF experiments. Increased CLMF performance was accompanied by a decrease in task latency and cortical dF/F0 amplitude during the task, indicating lower cortical activation as the task gets more familiar.

Relationships between measures of neurovascular integrity and fluid transport in aging: a multi-modal neuroimaging study
Fluid transport in the neurovascular unit (NVU) is essential for maintaining brain health through nutrient delivery and waste clearance. NVU integrity and fluid regulation can be assessed through MRI measures, including water exchange rate through the NVU (BBB kw), enlarged perivascular spaces (ePVS), cerebral blood flow (CBF), free water (FW), and white matter hyperintensities (WMH). This study investigated relationships between these MRI measures using Bayesian mixed models, and their variation with chronological age or biological brain age (brainageR) using linear regression in 132 non-clinical older adults (mean age=67 years; 68% female). BBB kw positively associated with CBF (^=0.08, 95% credible interval (CI)=[0.02,0.15]). FW positively associated with both ePVS (^=0.44, CI=[0.30,0.63]) and WMH (^=0.13, CI=[0.04,0.21]). BBB kw, CBF and ePVS decreased with age, while FW and WMH increased (all p<.05). There were no associations with brain age (all p>.05). Relationships between FW, ePVS and WMH likely reflect interconnectivity of fluid regulation within different compartments, while the relationship between BBB kw and CBF indicates a link between NVU fluid flow and vessel function. While individual metrics of NVU integrity are associated with age, their inter-relationships appear stable, providing a baseline for future research in fluid transport and vascular health in neurodegenerative disease.

Bidirectional control of neurovascular coupling by cortical somatostatin interneurons
Neurovascular coupling, which links neuronal activity to cerebral blood flow, is altered early in several neurological disorders and underlies functional brain imaging. This complex process involves multiple cellular players, with inhibitory interneurons in particular receiving increasing attention, yet the mechanisms underlying how they control of cerebral blood flow remain elusive. This study elucidates the mechanisms by which somatostatin interneurons bidirectionally control neurovascular coupling. Using patch-clamp recordings in cortical slices from mouse expressing channelrhodopsin-2 in somatostatin interneurons, we observed that these neurons are supralinearly activated by low-frequency (< 5 Hz) photostimulation and are efficiently and reliably activated at frequencies up to 20 Hz. Ex vivo vascular imaging showed that low-frequency (2 Hz) photostimulation triggered vasodilation whereas high-frequency (20 Hz) photostimulation induced vasoconstriction, both dependent on action potential firing. Histochemical analysis revealed that subpopulations of cortical somatostatin interneurons expressed NOS-1, the neuronal synthesizing enzyme of the vasodilator nitric oxide, and/or the vasoconstrictor neuropeptide Y at much greater extents. Consistently, pharmacological investigations have shown that vasodilation induced by low-frequency optogenetic stimulation involves nitric oxide release and activation of its vascular receptor soluble guanylate cyclase. In contrast, the vasoconstriction induced at high-frequency photostimulation involves neuropeptide Y release and activation of the Y1 vascular receptor. These findings provide valuable insights into neurovascular coupling and help to understand the cellular mechanism underlying the functional brain imaging signals used to map brain function in both health and disease.

Simultaneously induced slow and fast gamma waves travel independently in primate primary visual cortex
Travelling waves have been reported for multiple neuronal oscillations across the cortex. Within the primary visual cortex (V1), large visual stimuli (gratings) can induce two gamma oscillations, slow (20-35 Hz) and fast (40-60 Hz), potentially due to two different interneuronal classes. However, wave like behaviour of these rhythms and their relationship is unknown. We showed large gratings to monkeys that simultaneously induced slow and fast gamma while recording from V1 using microelectrode arrays. Both slow and fast gamma were organized as travelling waves that were not locked to stimulus onset. Direction of wave propagation was significantly different but showed no correlation, even in concurrent waves. Slow gamma waves lasted for longer durations than fast gamma. Wave direction varied with stimulus orientation and spatial frequency. Thus, slow and fast gamma oscillations in V1 show unique spatial and temporal wave dynamics signifying that these gamma rhythms are generated by distinct neural circuits.

Optimization of microRNA Target Sequence Arrangement in AAV Vectors Dramatically Enhances Specificity and Efficiency of Transgene Expression in Cortical Microglia
Microglia play a critical role in diseases such as Alzheimer's and stroke, making them a significant target for therapeutic intervention. However, due to their immune functions in detecting and combating viral invasion, efficient gene delivery to microglia remains challenging. We achieved specific and efficient gene delivery to microglia using an adeno-associated virus (AAV) vector designed for this purpose. This microglia-targeting AAV vector includes the mouse microglia/macrophage-specific ionized calcium-binding adaptor molecule 1 (mIba1) promoter, green fluorescent protein (GFP), microRNA target sequences (miR.Ts), woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and polyadenylation (polyA) signal, positioned between inverted terminal repeats. When miR.Ts were placed downstream of WPRE (between WPRE and polyA), gene expression occurred not only in microglia but also in a substantial number of neurons. However, when miR.Ts were positioned upstream of WPRE (between GFP and WPRE) or on both sides of WPRE, neuronal expression was significantly suppressed, resulting in selective GFP expression in microglia. Notably, positioning miR.Ts on both sides of WPRE achieved over 90% specificity and more than 60% efficiency in transgene expression in microglia three weeks after viral administration. This vector also enabled GCaMP expression in microglia, facilitating real-time monitoring of calcium dynamics and microglial process activity in the cortex. Additionally, intravenous administration of this vector with the blood-brain barrier-penetrant AAV-9P31 capsid variant resulted in extensive GFP expression selectively in microglia throughout the brain. These findings establish this AAV vector system as a robust tool for long-term, specific, and efficient gene expression in microglia.