bioRxiv Subject Collection: Neuroscience's Journal

Cell non-autonomous signaling through the conserved C. elegans glycopeptide hormone receptor FSHR-1 regulates cholinergic neurotransmission
Modulation of neurotransmission is key for organismal responses to varying physiological contexts such as during infection, injury, or other stresses, as well as in learning and memory and for sensory adaptation. Roles for cell autonomous neuromodulatory mechanisms in these processes have been well described. The importance of cell non-autonomous pathways for inter-tissue signaling, such as gut-to-brain or glia-to-neuron, has emerged more recently, but the cellular mechanisms mediating such regulation remain comparatively unexplored. Glycoproteins and their G protein-coupled receptors (GPCRs) are well-established orchestrators of multi-tissue signaling events that govern diverse physiological processes through both cell-autonomous and cell non-autonomous regulation. Here, we show that follicle stimulating hormone receptor, FSHR-1, the sole Caenorhabditis elegans ortholog of mammalian glycoprotein hormone GPCRs, is important for cell non-autonomous modulation of synaptic transmission. Inhibition of fshr-1 expression reduces muscle contraction and leads to synaptic vesicle accumulation in cholinergic motor neurons. The neuromuscular and locomotor defects in fshr-1 loss-of-function mutants are associated with an underlying accumulation of synaptic vesicles, build-up of the synaptic vesicle priming factor UNC-10/RIM, and decreased synaptic vesicle release from cholinergic motor neurons. Restoration of FSHR-1 to the intestine is sufficient to restore neuromuscular activity and synaptic vesicle localization to fshr-1-deficient animals. Intestine-specific knockdown of FSHR-1 reduces neuromuscular function, indicating FSHR-1 is both necessary and sufficient in the intestine for its neuromuscular effects. Re-expression of FSHR-1 in other sites of endogenous expression, including glial cells and neurons, also restored some neuromuscular deficits, indicating potential cross-tissue regulation from these tissues as well. Genetic interaction studies provide evidence that downstream effectors gsa-1/GalphaS, acy-1/adenylyl cyclase and sphk-1/sphingosine kinase and glycoprotein hormone subunit orthologs, GPLA-1/GPA2 and GPLB-1/GPB5, are important for FSHR-1 modulation of the NMJ. Together, our results demonstrate that FSHR-1 modulation directs inter-tissue signaling systems, which promote synaptic vesicle release at neuromuscular synapses.

Aligning with the predecessors and counterarguments: A systematic review of the anatomical correlates for the newly discovered meningeal layer in the existing literature
A recent study reported the existence of a subarachnoid lymphatic-like membrane (SLYM), an intermediate leptomeningeal layer between the arachnoid and pia mater in mice and human brains, dividing the subarachnoid space (SAS) into two functional compartments. Despite being a macroscopic structure, how it missed detection in previous studies is surprising. We systematically reviewed the published reports in animals and humans to explore whether prior descriptions of this meningeal layer have existed. An electronic search was conducted in PubMed/Medline, EMBASE, Google Scholar, Science Direct, and Web of Science databases using combinations of MeSH terms and keywords with Boolean operators from inception until 31st Dec 2023. We found at least eight studies that provided structural evidence of an intermediate leptomeningeal layer in the brain or spinal cord. However, unequivocal descriptions for this layer all along the central nervous system were scarce. Obscured names were used to describe it, i.e., the epipial layer, intermediate meningeal layer, intermediate lamella, and outer pial layer. Its microscopic/ultrastructural details closely resembled the SLYM. Further, we examined the counterarguments in current literature that are skeptical of this layer's existence. Considering the significant physiological/clinical implications, exploring further structural and functional details of the new meningeal layer is a need of the hour.

Functional dynamics and selectivity of two parallel corticocortical pathways from motor cortex to layer 5 circuits in somatosensory cortex
Long-range corticocortical pathways mediate direct interactions between the primary motor cortex (M1) and the somatosensory cortex (S1) and are likely critical for context-dependent sensory processing and sensorimotor integration. In the rodent whisker system, projections from M1 to S1 may be necessary for interpreting touch signals in the context of ongoing movement to drive behavior. However, understanding the function of these interareal interactions requires knowledge about the physiological properties of the synapses themselves and how specific classes of neurons integrate those signals. Here, we combined optogenetics and retrograde labeling with in vitro electrophysiology to characterize the synaptic properties of the connections between M1 and layer 5 (L5) intratelencephalic (IT) and pyramidal tract (PT) neurons in S1 of the mouse (both sexes) and how these two classes of neurons integrate those inputs. We found that M1 excitatory inputs to L5 IT cells depressed but had slow time courses that resulted in summation at the soma, whereas inputs to L5 PT cells facilitated and had faster time courses, resulting in less temporal summation at the soma. Differences in hyperpolarization-activated current (Ih) could partially explain the differences in subthreshold synaptic responses between L5 neurons. Functionally, we found that high-frequency M1 activity coupled more effectively with backpropagating action potentials within a narrow time window in PT neurons to trigger bursts at the soma. Our findings highlight the synaptic and cellular dynamics of two parallel pathways underlying the interactions between M1 and specific L5 circuits in S1.

Dose escalation pre-clinical trial of novel Dok7-AAV in mouse model of DOK7 congenital myasthenia
Congenital myasthenic syndromes (CMS) are a group of inherited disorders characterised by defective neuromuscular transmission and fatigable muscle weakness. Mutations in DOK7, a gene encoding a post-synaptic protein crucial in the formation and stabilisation of the neuromuscular junction (NMJ), rank among the leading three prevalent causes of CMS in diverse populations globally. The majority of DOK7 CMS patients experience varying degrees of disability despite receiving optimised treatment, necessitating the development of improved therapeutic approaches. Here we executed a dose escalation pre-clinical trial using a DOK7-CMS mouse model to assess the efficacy of Amp-101, an innovative AAV gene replacement therapy. Amp-101 is based on AAVrh74 and contains human DOK7 cDNA under the control of a muscle-restricted promoter. We show that at doses 6x1013vg/kg and 1x1014 vg/kg, Amp-101 generated enlarged NMJs and rescued the very severe phenotype of the model. Treated mice became at least as strong as WT littermates and the diaphragm and tibialis anterior muscles displayed robust expression of DOK7. This data suggests that Amp-101 is a promising candidate to move forward to clinic trials.

Impaired drainage through capillary-venous networks contributes to age-related white matter loss
The gradual loss of cerebral white matter contributes to cognitive decline during aging. However, microvascular networks that support the metabolic demands of white matter remain poorly defined. We used in vivo deep multi-photon imaging to characterize microvascular networks that perfuse cortical layer 6 and corpus callosum, a highly studied region of white matter in the mouse brain. We show that these deep tissues are exclusively drained by sparse and wide-reaching venules, termed principal cortical venules, which mirror vascular architecture at the human cortical-U fiber interface. During aging, capillary networks draining into deep branches of principal cortical venules are selectively constricted, reduced in density, and diminished in pericyte numbers. This causes hypo-perfusion in deep tissues, and correlates with gliosis and demyelination, whereas superficial tissues become relatively hyper-perfused. Thus, age-related impairment of capillary-venular drainage is a key vascular deficit that contributes to the unique vulnerability of cerebral white matter during brain aging.

Asymmetric representation of symmetric semantic information in the human brain
Specific pairs of semantic entities have symmetric relationships, such as word pairs with opposite meanings (e.g., "intelligent" and "stupid"; "human" and "mechanical"). Such semantic symmetry is a key feature of semantic information. However, the representation of symmetric semantic information in the brain is not yet understood. Additionally, it is unclear whether symmetric pairs of semantic information do have symmetric representations in the brain? We addressed this question in a data-driven manner by using the voxelwise modeling of movie-evoked cortical response measured by functional magnetic resonance imaging. In this modeling, response in each voxel was predicted from semantic labels designated for each movie scene. The semantic labels consisted of 30 different items, including 15 pairs of semantically symmetric items. Each item was manually evaluated using a 5-point scale. By localizing the semantic representation associated with each item based on the voxelwise accuracy of brain-response predictions, we found that semantic representations of symmetric item pairs are broadly distributed but with little overlap in the cortex. Additionally, the weight of voxelwise models revealed highly complex, various patterns of cortical representations for each item pair. These results suggest that symmetric semantic information is rather asymmetric and heterogeneous representations in the human brain.

Brain activation during the N-back working memory task in individuals with spinal cord injury: a functional near-infrared spectroscopy study
Cognitive impairments have frequently been reported in individuals with spinal cord injury (SCI) across different domains such as working memory, attention, and executive function. The mechanism of cognitive impairment after SCI is not well understood due to the heterogeneity of SCI sample populations, and may possibly be due to factors such as cardiovascular dysfunction, concomitant traumatic brain injury (TBI), hypoxia, sleep disorders, and body temperature dysregulation. In this study, we implement the Neuropsychiatric Unit Cognitive Assessment Tool (NUCOG) to assess cognitive differences between individuals with SCI and age-matched able-bodied (AB) controls. We then use an N-back working memory task and functional near-infrared spectroscopy (fNIRS) to elucidate the neurovascular correlates of cognitive function in individuals with SCI. We observed significant differences between the SCI and AB groups on measures of executive function on the NUCOG test. On the N-back task, across the three levels of difficulty: 0-back, 2-back, and 3-back, no significant differences were observed between the SCI and AB group; however, both groups performed worse as the level of difficulty increased. Although there were no significant differences in N-back performance scores between the two groups, functional brain hemodynamic activity differences were observed between the SCI and AB groups, with the SCI group exhibiting higher maximum oxygenated hemoglobin concentration in the right inferior parietal lobe. These findings support the use of fNIRS to study cognitive function in individuals with SCI and may provide a useful tool during rehabilitation to obtain quantitative functional brain activity metrics.

Automated multimodal imaging of Caenorhabditis elegans behavior in multi-well plates
Assays of behavior in model organisms play an important role in genetic screens, drug testing, and the elucidation of gene-behavior relationships. We have developed an automated, high-throughput imaging and analysis method for assaying behaviors of the nematode C. elegans. We use high-resolution optical imaging to longitudinally record the behaviors of 96 animals at a time in multi-well plates, and computer vision software to quantify the animals' locomotor activity, behavioral states, and egg laying events. To demonstrate the capabilities of our system we used it to examine the role of serotonin in C. elegans behavior. We found that egg-laying events are preceded by a period of reduced locomotion, and that this decline in movement requires serotonin signaling. In addition, we identified novel roles of serotonin receptors SER-1 and SER-7 in regulating the effects of serotonin on egg laying across roaming, dwelling, and quiescent locomotor states. Our system will be useful for performing genetic or chemical screens for modulators of behavior.

Closed-loop electrical stimulation to prevent focal epilepsy progression and long-term memory impairment
Interictal epileptiform discharges (IEDs) are ubiquitously expressed in epileptic networks and disrupt cognitive functions. It is unclear whether addressing IED-induced dysfunction could improve epilepsy outcomes as most therapeutics target seizures. We show in a model of progressive hippocampal epilepsy that IEDs produce pathological oscillatory coupling which is associated with prolonged, hypersynchronous neural spiking in synaptically connected cortex and expands the brain territory capable of generating IEDs. A similar relationship between IED-mediated oscillatory coupling and temporal organization of IEDs across brain regions was identified in human subjects with refractory focal epilepsy. Spatiotemporally targeted closed-loop electrical stimulation triggered on hippocampal IED occurrence eliminated the abnormal cortical activity patterns, preventing spread of the epileptic network and ameliorating long-term spatial memory deficits in rodents. These findings suggest that stimulation-based network interventions that normalize interictal dynamics may be an effective treatment of epilepsy and its comorbidities, with a low barrier to clinical translation.

Impaired 26S proteasome causes leaning and memory deficiency and induces neuroinflammation mediated by NF-kappaB in mice
Aging and many neurodegenerative disorders, including Alzheimer disease (AD), show reduced proteasome activity, loss of synapses and increased neuroinflammation in the brain. However, whether proteasome dysfunction causes neuroinflammation remains less understood. Here, we studied the effect of impaired 26S proteasome on neuroinflammation in the Psmc1 knockout (KO) mice deficient of a 19S proteasome subunit selectively in the forebrain region. We initially assessed the proteasome activity in the KO and control mouse brains and performed cognitive behavioral test to the animals. The synaptosomes were isolated from the forebrain and subjected to mass spectrometric analysis of total synaptic proteins. The identified synaptic proteins were then assessed using bioinformatic approaches. To validate identified proteins, Western blot analysis and immunofluorescent staining of specific protein were utilized. To validate NF-kB mediating neuroinflammation, we administered the KO animals at 5 weeks of age with a selective NF-kB inhibitor for three weeks and then, animal behaviors and neuroinflammation were assessed when they reached to eight weeks of age. Our results revealed that impaired 26S proteasome led to AD-like cognitive deficiency and overt neuroinflammation in the synapses of the Psmc1 KO brain at eight weeks of age. Pronounced neuroinflammation also occurred in other regions and glial cells, which was confirmed by increased levels of several key immune response-related proteins, including Stat1, Trem2 and NF-kB, and by activation of astrocytes and microglia in the KO brain. Following treating the KO mice with a selective NF-kB inhibitor, the KO mice exhibited improved behaviors and reduced neuroinflammation compared to the control animals. These data indicate that impaired 26S proteasome causes AD-like cognitive deficiency and induces neuroinflammation mediated largely by NF-kB, which will help develop effective therapeutics and aid to better understand the pathogenesis of AD and many other neurodegenerative disorders where impaired proteasome is consistently coupled with neuroinflammation.

Mind the blank: behavioral, experiential, and physiological signatures of absent-mindedness
Does being awake mean being conscious? This study investigates Mind Blanking (MB), characterized by an "emptiness of mind", comparing it with Mind Wandering (MW) and On-task (ON) states. Using a sustained attention task and electroencephalogram monitoring on 26 participants, behavioral and neurophysiological signatures of MB were examined. MB exhibited a specific pattern of behavioral lapses, as well as decreased fast oscillatory activity and complexity over posterior electrodes compared to MW. Functional connectivity analyses also revealed decreased frontal-posterior connectivity during MB and event-related potentials indicated a disruption in late-stage visual processing, suggesting a lack of conscious access to sensory information during MB. EEG-based neural features enabled trial-level prediction of mental states, furnishing estimates of the fine-grained dynamics of consciousness beyond subjective reports. Overall, these findings challenge the notion of continuous wake consciousness, suggesting that MB represents genuine gaps in our stream of thoughts.

Increased excitability of dentate gyrus mossy cells occurs early in life in the Tg2576 model of Alzheimer's disease.
INTRODUCTION: Hyperexcitability in Alzheimer's disease (AD) emerge early and contribute to disease progression. The dentate gyrus (DG) is implicated in hyperexcitability in AD. We hypothesized that mossy cells (MCs), regulators of DG excitability, contribute to early hyperexcitability in AD. Indeed, MCs generate hyperexcitability in epilepsy. METHODS: Using the Tg2576 model and WT mice (~1month-old), we compared MCs electrophysiologically, assessed c-Fos activity marker, A{beta} expression and mice performance in a hippocampal-dependent memory task. RESULTS: Tg2576 MCs exhibit increased spontaneous excitatory events and decreased inhibitory currents, increasing the charge transfer excitation/inhibition ratio. Tg2576 MC intrinsic excitability was enhanced, and showed higher c-Fos, intracellular A{beta} expression, and axon sprouting. Granule cells only showed changes in synaptic properties, without intrinsic changes. The effects occurred before a memory task is affected. DISCUSSION: Early electrophysiological and morphological alterations in Tg2576 MCs are consistent with enhanced excitability, suggesting an early role in DG hyperexcitability and AD pathophysiology.

Alterations in Lysosomal, Glial and Neurodegenerative Biomarkers in Patients with Sporadic and Genetic Forms of Frontotemporal Dementia
Frontotemporal dementia (FTD) is the most common cause of early-onset dementia with 10-20% of cases caused by mutations in one of three genes: GRN, C9orf72, or MAPT. To effectively develop therapeutics for FTD, the identification and characterization of biomarkers to understand disease pathogenesis and evaluate the impact of specific therapeutic strategies on the target biology as well as the underlying disease pathology are essential. Moreover, tracking the longitudinal changes of these biomarkers throughout disease progression is crucial to discern their correlation with clinical manifestations for potential prognostic usage. Methods We conducted a comprehensive investigation of biomarkers indicative of lysosomal biology, glial cell activation, synaptic and neuronal health in cerebrospinal fluid (CSF) and plasma from non-carrier controls, sporadic FTD (symptomatic non-carriers) and symptomatic carriers of mutations in GRN, C9orf72, or MAPT, as well as asymptomatic GRN mutation carriers. We also assessed the longitudinal changes of biomarkers in GRN mutation carriers. Furthermore, we examined biomarker levels in disease impacted brain regions including middle temporal gyrus (MTG) and superior frontal gyrus (SFG) and disease-unaffected inferior occipital gyrus (IOG) from sporadic FTD and symptomatic GRN carriers. Results We confirmed glucosylsphingosine (GlcSph), a lysosomal biomarker regulated by progranulin, was elevated in the plasma from GRN mutation carriers, both symptomatic and asymptomatic. GlcSph and other lysosomal biomarkers such as ganglioside GM2 and globoside GB3 were increased in the disease affected SFG and MTG regions from sporadic FTD and symptomatic GRN mutation carriers, but not in the IOG, compared to the same brain regions from controls. The glial biomarkers GFAP in plasma and YKL40 in CSF were elevated in asymptomatic GRN carriers, and all symptomatic groups, except the symptomatic C9orf72 mutation group. YKL40 was also increased in SFG and MTG regions from sporadic FTD and symptomatic GRN mutation carriers. Neuronal injury and degeneration biomarkers NfL in CSF and plasma, and UCHL1 in CSF were elevated in patients with all forms of FTD. Synaptic biomarkers NPTXR, NPTX1/2, and VGF were reduced in CSF from patients with all forms of FTD, with the most pronounced reductions observed in symptomatic MAPT mutation carriers. Furthermore, we demonstrated plasma NfL was significantly positively correlated with disease severity as measured by CDR+NACC FTLD-SB in genetic forms of FTD and CSF NPTXR was significantly negatively correlated with CDR+NACC FTLD-SB in symptomatic GRN and MAPT mutation carriers. Conclusions In conclusion, our comprehensive investigation replicated alterations in biofluid biomarkers indicative of lysosomal function, glial activation, synaptic and neuronal health across sporadic and genetic forms of FTD and unveiled novel insights into the dysregulation of these biomarkers within brain tissues from patients with GRN mutations. The observed correlations between biomarkers and disease severity open promising avenues for prognostic applications and for indicators of drug efficacy in clinical trials. Our data also implicated a complicated relationship between biofluid and tissue biomarker changes and future investigations should delve into the mechanistic underpinnings of these biomarkers, which will serve as a foundation for the development of targeted therapeutics for FTD.

Benchmark of cellular deconvolution methods using a multi-assay reference dataset from postmortem human prefrontal cortex
Background: Cellular deconvolution of bulk RNA-sequencing (RNA-seq) data using single cell or nuclei RNA-seq (sc/snRNA-seq) reference data is an important strategy for estimating cell type composition in heterogeneous tissues, such as human brain. Several deconvolution methods have been developed and they have been previously benchmarked against simulated data, pseudobulked sc/snRNA-seq data, or cell type proportions derived from immunohistochemistry reference data. A major limitation preventing the improvement of deconvolution algorithms has been the lack of highly integrated datasets with orthogonal measurements of gene expression and estimates of cell type proportions on the same tissue block. The performance of existing deconvolution algorithms has not yet been explored across different RNA extraction methods (e.g. cytosolic, nuclear, or whole cell RNA), different library preparation types (e.g. mRNA enrichment vs. ribosomal RNA depletion), or with matched single cell reference datasets. Results: A rich multi-assay dataset was generated in postmortem human dorsolateral prefrontal cortex (DLPFC) from 22 tissue blocks. Assays included spatially-resolved transcriptomics, snRNA-seq, bulk RNA-seq across six RNA extraction and RNA-seq library combinations, and orthogonal cell type measurements via RNAScope/Immunofluorescence (RNAScope/IF). The Mean Ratio method was developed for selecting cell type marker genes for deconvolution and is implemented in the DeconvoBuddies R package. Five extensively benchmarked computational deconvolution algorithms were evaluated in DLPFC across six RNA-seq combinations and predicted cell type proportions were compared to those measured by RNAScope/IF. Conclusions: We show that Bisque and hspe are the top performing methods with performance dependent on the RNA-seq library preparation conditions. We provide a multi-assay resource for the development and evaluation of deconvolution algorithms.

Cyclin-dependent kinase 5 (Cdk5) activity is modulated by light and gates rapid phase shifts of the circadian clock
Changes in lighting conditions as they occur naturally over seasons or manmade by jet lag or shift work, advance or delay clock phase to synchronize physiology to the environment. Within the suprachiasmatic nucleus (SCN) of the hypothalamus, circadian timekeeping and resetting have been shown to depend on both membrane depolarization and intracellular second-messenger signaling. In both processes, voltage-gated calcium channels (VGCCs) mediate calcium influx resulting in the activation of intracellular signaling pathways that activate Period (Per) gene expression. However, the precise mechanism how these processes are gated in a concerted manner is unknown. Here we show that Cdk5 activity is modulated by light and gates phase shifts of the circadian clock. We found that knock-down of Cdk5 in the SCN of mice affects phase delays but not phase advances. This is associated with uncontrolled calcium influx into SCN neurons and an unregulated PKA - CaMK - CREB signaling pathway. Our experiments identified an important light modulated kinase that affects rapid clock phase adaptation, which is important to adapt activity onset to seasonal changes, jet-lag and shift work.

Novel murine closed-loop auditory stimulation paradigm elicits macrostructural sleep benefits in neurodegeneration
Boosting slow-wave activity (SWA) by modulating slow-waves through closed-loop auditory stimulation (CLAS) might provide a powerful nonpharmacological tool to investigate the link between sleep and neurodegeneration. Nevertheless, CLAS in this context was not yet explored. Here, we established mouse CLAS (mCLAS)-mediated SWA enhancement and explored its effects onto sleep deficits in neurodegeneration, by targeting the up-phase of slow-waves in mouse models of Alzheimer's (AD, Tg2576) and Parkinson's disease (PD, M83). We found that tracking a 2Hz component of slow-waves leads to highest precision of NREM sleep detection in mice, and that its combination with a 30 degree up-phase-target produces a significant SWA 15-30% increase from baseline in WTAD and TGAD mice versus a MOCK group. Conversely, combining 2Hz with a 40 degree phase target yields a significant increase ranging 30-35% in WTPD and TGPD mice. Interestingly, these phase-target-triggered SWA increases are not genotype dependent but strain specific. Sleep alterations that may contribute to disease progression and burden were described in AD and PD lines. Notably, pathological sleep traits where rescued by mCLAS, which elicited a 14% decrease of pathologically heightened NREM sleep fragmentation in TGAD mice, accompanied by a steep decrease in microarousal events during both light and dark periods. Overall, our results indicate that model-tailored phase-targeting is key to modulate SWA through mCLAS, prompting the acute alleviation of key neurodegeneration-associated sleep phenotypes and potentiating sleep regulation and consolidation. Further experiments assessing the long-term effect of mCLAS in neurodegeneration may majorly impact the establishment of sleep-based therapies.

Supramodal neural information supports stimulus-driven attention across cortical levels
Sensory systems utilise stimulus-driven attention to survey the environment for significant features. The question arises: are the cortical networks that influence stimulus-driven attention supramodal or specific to each sensory modality? Here we employed a hierarchical target detection task (n=30), examining cortical responses linked to the detection of salient targets in the somatosensory and auditory modality. In a temporal decoding analysis, we reveal a transient early supramodal process activated by target detection. We also demonstrate that both common and unique modulations of salience-related cortical responses to somatosensory and auditory targets involve modality-specific and frontal regions using Parametric Empirical Bayes. Specifically, we found that the inferior frontal gyri share information across both sensory modalities, while recurrent information transfer between ipsilateral inferior frontal gyri and associative regions was modality-specific. Finally, we showed both supramodal and modality-specific attentional modulations of effective connectivity linking regions across hierarchical levels in the cortex. Our results provide evidence for an attention network which integrates information across inferior frontal cortices to detect salient targets irrespective of their specific sensory modality. Beyond the notion of a supramodal attention system, our findings support the role of modality-specific cortices in processing inputs from other sensory modalities, highlighting that attention can bias these processes at multiple stages of the cortical hierarchy.

Influence of chondroitin sulfate glycan sulfation patterns on histochemical labeling of perineuronal nets- a comparative study of interregional distribution in human and mouse brain
Perineuronal nets (PNNs) are a condensed subtype of extracellular matrix that form a net-like coverings around certain neurons in the brain. PNNs are primarily composed of chondroitin sulfate (CS) proteoglycans from the lectican family that consist of CS-glycosaminoglycan (CS-GAG) side chains attached to a core protein. CS disaccharides (CS-d) can exist in various isoforms with different sulfation patterns. Literature suggests that CS-d sulfation patterns can influence the function of PNNs as well as their labeling. This study was conducted to characterize such interregional CS-d sulfation pattern differences in adult human (N = 81) and mouse (N = 19) brains. Liquid chromatography tandem mass spectrometry was used to quantify five different CS-d sulfation patterns, which were then compared to immunolabeling of PNNs using Wisteria Floribunda Lectin (WFL) to identify CS-GAGs and anti-aggrecan to identify CS proteoglycans. In healthy brains, significant regional and species-specific differences in CS-d sulfation and single versus double-labeling pattern were identified. A secondary analysis to investigate how early-life stress (ELS) impacts these PNN features discovered that although ELS increases WFL+ PNN density, the CS-GAG sulfation code and single versus double PNN-labeling distributions remained unaffected in both species. These results underscore PNN complexity in traditional research, emphasizing the need to consider their heterogeneity in future experiments.

Dorsal Raphe to Basolateral Amygdala Corticotropin-Releasing Factor Circuit Regulates Cocaine-Memory Reconsolidation
Environmental stimuli elicit drug craving and relapse in cocaine users by triggering the retrieval of strong cocaine-related contextual memories. Retrieval can also destabilize drug memories, requiring reconsolidation, a protein synthesis-dependent storage process, to maintain memory strength. Corticotropin-releasing factor (CRF) signaling in the basolateral amygdala (BLA) is necessary for cocaine-memory reconsolidation. We have hypothesized that a critical source of CRF in the BLA is the dorsal raphe nucleus (DR) based on its neurochemistry, anatomical connectivity, and requisite involvement in cocaine-memory reconsolidation. To test this hypothesis, male and female Sprague-Dawley rats received adeno-associated viruses to express Gi-coupled designer receptors exclusively activated by designer drugs (DREADDs) selectively in CRF neurons of the DR and injection cannulae directed at the BLA. The rats were trained to self-administer cocaine in a distinct environmental context then received extinction training in a different context. They were then briefly re-exposed to the cocaine-predictive context to destabilize (reactivate) cocaine memories. Intra-BLA infusions of the DREADD agonist deschloroclozapine (DCZ; 0.1 mM, 0.5 L/hemisphere) after memory reactivation attenuated cocaine-memory strength, relative to vehicle infusion. This was indicated by a selective, DCZ-induced and memory reactivation-dependent decrease in drug-seeking behavior in the cocaine-predictive context in DREADD-expressing males and females at test compared to respective controls. Notably, BLA-projecting DR CRF neurons that exhibited increased c-Fos expression during memory reconsolidation co-expressed glutamatergic and serotonergic neuronal markers. Together, these findings suggest that the DRCRF [->] BLA circuit is engaged to maintain cocaine-memory strength after memory destabilization, and this phenomenon may be mediated by DR CRF, glutamate, and/or serotonin release in the BLA.

Influence of insulin sensitivity on food cue evoked functional brain connectivity in children
Objective: Insulin resistance during childhood is a risk factor for developing type 2 diabetes and other health problems later in life. Studies in adults have shown that insulin resistance affects regional and network activity in the brain which are vital for behavior, e.g. ingestion and metabolic control. To date, no study has investigated whether brain responses to food cues in children are associated with peripheral insulin sensitivity. Methods: We included 53 children (36 girls) between the age of 7-11 years, who underwent an oral Glucose Tolerance Test (oGTT) to estimate peripheral insulin sensitivity (ISI). Brain responses were measured using functional magnetic resonance imaging (fMRI) before and after glucose ingestion. We compared food-cue task-based activity and functional connectivity (FC) between children with low and high ISI, adjusted for age and BMIz. Results: Independent of prandial state (i.e., glucose ingestion), children with lower ISI showed higher FC between the anterior insula and caudate and lower FC between the posterior insula and mid temporal cortex than children with higher ISI. Sex differences were found based on prandial state and peripheral insulin sensitivity in the insular FC. No differences were found on whole-brain food-cue reactivity. Conclusions: Children with low peripheral insulin sensitivity showed differences in food cue evoked response particularly in insula functional connectivity. These differences might influence eating behavior and future risk of developing diabetes.

Extracellular disposal of nuclear waste by APP: a protective mechanism impaired in Alzheimer's disease
Although the amyloid beta (Abeta) hypothesis (1) has long been central to Alzheimer's disease (AD) research, effective therapeutic strategies remain elusive (2,3). Here we re-evaluate the functions of amyloid precursor protein (APP) and reveal its critical function in protecting against nuclear impairment-induced cell death and inflammation (4,5). Overexpression of APP mitigated etoposide or lamin A knockdown-induced nuclear damage, while APP removal or mutations exacerbated these effects. Interestingly, neurons differentiated from induced pluripotent stem cells (iPSCs) exhibited similar patterns, and notably, familial AD-associated mutant APP failed to confer protection against nuclear impairment. We identify APP's interaction with a cytoplasmic structure of nuclear origin, termed 'nuclear waste', and propose its role in extracellular waste disposal. Intriguingly, cells lacking APP showed impaired nuclear waste clearance, leading to abnormal cytoplasmic accumulation of the nuclear waste. Similarly, neuron-specific APP overexpression using adeno-associated virus (AAV) in mice reduced neuronal death and inflammation caused by nuclear damage. Conversely, shRNA-mediated APP exacerbated these effects, and mutant APP associated with familial AD lacked protective effects. Moreover, postmortem analysis of AD brains revealed accumulation of abnormal nuclear waste in the neurocytoplasm, irregular nuclear morphology, and reduced APP levels per neuron. Our data underscore APP's crucial role in disposing of nuclear waste, maintaining cellular homeostasis, and suggest its dysregulation as a potential contributor to AD pathogenesis. Restoring APP waste clearance in AD could be a promising target for disease-modifying therapies.

In mice, discrete odors can selectively promote the neurogenesis of sensory neuron subtypes that they stimulate
In mammals, olfactory sensory neurons (OSNs) are born throughout life, presumably solely to replace neurons lost via turnover or injury. This assumption follows from the hypothesis that olfactory neurogenesis is strictly stochastic with respect to neuron subtype, as defined by the single odorant receptor allele that each neural precursor stochastically chooses out of hundreds of possibilities. This hypothesis is challenged by recent findings that the birthrates of a fraction of subtypes are selectively diminished by olfactory deprivation. These findings raise questions about how, and why, olfactory stimuli are required to promote the neurogenesis of some OSN subtypes, including whether the stimuli are generic (e.g., broadly activating odors or mechanical stimuli) or specific (e.g., discrete odorants). Based on RNA-seq and scRNA-seq analyses, we hypothesized that the neurogenic stimuli are specific odorants that selectively activate the same OSN subtypes whose birthrates are accelerated. In support of this, we have found, using subtype-specific OSN birthdating, that exposure to male and musk odors can accelerate the birthrates of responsive OSNs. Collectively, our findings reveal that certain odor experiences can selectively "amplify" specific OSN subtypes, and that persistent OSN neurogenesis may serve, in part, an adaptive function.

Chemogenetic activation of microglial Gi signaling decreases microglial surveillance and impairs neuronal synchronization
Microglia actively survey the brain and dynamically interact with neurons to maintain brain homeostasis. Microglial Gi-protein coupled receptors (Gi-GPCRs) play a critical role in microglia-neuron communications. However, the impact of temporally activating microglial Gi signaling on microglial dynamics and neuronal activity in the homeostatic brain remains largely unknown. In this study, we employed Gi-based Designer Receptors Exclusively Activated by Designer Drugs (Gi-DREADD) to selectively and temporally modulate microglial Gi signaling pathway. By integrating this chemogenetic approach with in vivo two-photon imaging, we observed that exogenous activation of microglial Gi signaling transiently inhibited microglial process dynamics, reduced neuronal activity, and impaired neuronal synchronization. These altered neuronal functions were associated with a decrease in interactions between microglia and neuron somata. Altogether, this study demonstrates that acute, exogenous activation of microglial Gi signaling can regulate neuronal circuit function, offering a potential pharmacological target for neuromodulation through microglia.

Modeling Hereditary Diffuse Leukoencephalopathy with Axonal Spheroids using microglia-sufficient brain organoids
Hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS) is a rare, fatal, adult-onset neurodegenerative disease that is most often caused by mutations affecting the Colony Stimulating factor-1 Receptor (CSF-1R). To understand how CSF-1R-mutation affects human microglia - the specialized brain-resident macrophages of the central nervous system - and the downstream consequences for neuronal cells, we used a macrophage and forebrain organoid co-culture system based on induced pluripotent stem cells generated from two patients with HDLS, with CSF-1R gene-corrected isogenic organoids as controls. Macrophages derived from iPSC (iMacs) of patients exhibited a metabolic shift towards the glycolytic pathway and reduced CSF-1 sensitivity, which was associated with higher levels of IL-1{beta}production and an activated inflammatory phenotype. Single-cell RNA sequencing revealed that iMacs adopt a reactive state that leads to impaired regulation of neuronal cell populations in organoid cultures, thereby identifying microglial dysregulation and specifically IL-1{beta} production as key contributors to the degenerative neuro-environment in HDLS.

Two distinct mechanisms for Nav1.7 null analgesia
Genetic deletion and pharmacological inhibition are distinct approaches to unravelling pain mechanisms, identifying targets and developing new analgesics. Both approaches have been applied to the voltage-gated sodium channels Nav1.7 and Nav1.8. Genetic deletion of Nav1.8 in mice leads to a loss of pain, and antagonists are effective human analgesics. Complete embryonic loss of Nav1.7 in humans or in mouse sensory neurons leads to profound analgesia substantially mediated by endogenous opioid signaling, and anosmia that is opioid independent. Autonomic function appears to be normal. Adult deletion of Nav1.7 in sensory neurons also leads to analgesia with diminished sensory neuron excitability but there is no opioid component of analgesia. Pharmacological inhibition of Nav1.7 leads to dramatic side-effects on the autonomic nervous system. Here we compare and contrast the distinct embryonic and adult null mechanisms of Nav1.7 loss-of-function analgesia. We describe an endogenous opioid mechanism of analgesia that provides new opportunities for therapeutic intervention and pain relief.

How to design optimal brain stimulation to modulate phase-amplitude coupling?
Objective. Phase-amplitude coupling (PAC), the coupling of the amplitude of a faster brain rhythm to the phase of a slower brain rhythm, plays a significant role in brain activity and has been implicated in various neurological disorders. For example, in Parkinson's disease, PAC between the beta (13-30 Hz) and gamma (50-200 Hz) rhythms in the motor cortex is exaggerated, while in Alzheimer's disease, PAC between the theta (4-8 Hz) and gamma rhythms is diminished. Modulating PAC (i.e. reducing or enhancing PAC) using brain stimulation could therefore open new therapeutic avenues. However, while it has been previously reported that phase-locked stimulation can increase PAC, it is unclear what the optimal stimulation strategy to modulate PAC might be. Here, we provide a theoretical framework to narrow down the experimental optimisation of stimulation aimed at modulating PAC, which would otherwise rely on trial and error. Approach. We make analytical predictions using a Stuart-Landau model, and confirm these predictions in a more realistic model of coupled neural populations. Main results. Our framework specifies the critical Fourier coefficients of the stimulation waveform which should be tuned to optimally modulate PAC. Depending on the characteristics of the amplitude response curve of the fast population, these components may include the slow frequency, the fast frequency, combinations of these, as well as their harmonics. We also show that the optimal balance of energy between these Fourier components depends on the relative strength of the endogenous slow and fast rhythms, and that the alignment of fast components with the fast rhythm should change throughout the slow cycle. Furthermore, we identify the conditions requiring to phase-lock stimulation to the fast and/or slow rhythms. Significance. Together, our theoretical framework lays the foundation for guiding the development of innovative and more effective brain stimulation aimed at modulating PAC for therapeutic benefit.

Amelioration of signalling deficits underlying metabolic shortfall in TREM2R47H human iPSC-derived microglia
Classic immunometabolic switching from oxidative phosphorylation (OXPHOS) to glycolysis occurs in myeloid cells such as microglia when encountering pathogenic insults. We previously showed that patient iPSC-derived microglia (iPS-Mg) harbouring the Alzheimer's disease (AD) TREM2R47H hypomorph display deficits in the ability to metabolically switch from OXPHOS to glycolysis, with both reduced mitochondrial maximal respiration and glycolytic capacity. To resolve this, we generated common variant, TREM2R47H and TREM2-/- variant human iPS-Mg and we assessed the ability of supplementation with citrate or succinate, key metabolites and cell cycle breaking points upon microglia activation, to overcome deficits in a number of functions we identified previously as affected by TREM2 loss of function phenotypes. Succinate supplementation was more effective than citrate at overcoming mitochondrial deficits in OXPHOS and did not promote a switch to glycolysis. Citrate enhanced the lipid content of TREM2R47H iPS-Mg and was more effective at overcoming phagocytic deficits whereas succinate increased lipid content and phagocytic capacity in TREM2-/- iPS-Mg. Microglia cytokine release upon activation was differentially affected by citrate or succinate and neither metabolite altered soluble TREM2 shedding. Neither citrate nor succinate enhanced glycolysis but drove their effects through oxidative phosphorylation. Our data point to discrete pathway linkage between microglial metabolism and functional outcomes with implications for AD pathogenesis and treatments.

Continuous Bump Attractor Networks Require Explicit Error Coding for Gain Recalibration
Internal representations of continuous variables are crucial to create internal models of the external world. A prevailing model of how brain maintains these representations is given by continuous bump attractor networks (CBANs). CBANs have been hypothesized as an underlying mechanism in a broad range of brain functions across different areas, such as spatial navigation in hippocampal/entorhinal circuits and working memory in prefrontal cortex. Through recurrent connections, a CBAN maintains a persistent activity bump, whose peak location can vary along a neural space, corresponding to different values of a continuous variable. To track the value of a continuous variable changing over time, a CBAN updates the location of its activity bump based on inputs that encode the changes in the continuous variable (e.g., movement velocity in the case of spatial navigation) -- a process akin to mathematical integration. This integration process is not perfect and accumulates error over time. For error correction, CBANs can use additional inputs providing ground-truth information about the continuous variable's correct value (e.g., visual landmarks for spatial navigation). These inputs enable the network dynamics to automatically correct any representation error by shifting the activity bump toward the correct location. Recent experimental work on hippocampal place cells has shown that, beyond correcting errors, ground-truth inputs (e.g., visual landmarks) also fine-tune the gain of the integration process, a crucial factor that links the change in the continuous variable (e.g., movement velocity) to the updating of the activity bump's location. However, existing CBAN models lack this plasticity, offering no insights into the neural mechanisms and representations involved in the recalibration of the integration gain. In this paper, we explore this gap by using a ring attractor network, a specific type of CBAN, to model the experimental conditions that demonstrated gain recalibration in hippocampal place cells. Our analysis reveals the neural mechanisms behind gain recalibration within a CBAN: Unlike error correction, which occurs through network dynamics based on ground-truth inputs, gain recalibration requires an additional neural signal that explicitly encodes the error in the network's representation. This error signal must be provided by some neurons whose firing rate varies monotonically with respect to one of two signals---either the instantaneous error or the time-integral of the error---for recalibration of the integration gain. Finally, we propose a modified ring attractor network as an example CBAN model that verifies our theoretical findings. Combining an error-rate code with Hebbian synaptic plasticity, this model achieves recalibration of integration gain in a CBAN, ensuring accurate representation for continuous variables.

The impact of face masks on face-to-face neural tracking of speech: auditory and visual obstacles
Face masks provide fundamental protection against the transmission of respiratory viruses but hamper communication. We estimated auditory and visual obstacles generated by face masks on communication by measuring the neural tracking of face-to-face speech. To this end, we recorded the EEG while participants were exposed to naturalistic audio-visual speech, embedded in multi-talker noise, in three contexts: (i) no-mask (audio-visual information was fully available), (ii) virtual mask (occluded lips, but intact audio), and (iii) real mask (occluded lips and degraded audio). The neural tracking of lip movements and the sound envelope of speech was measured through backward modeling, that is, by reconstructing stimulus properties from neural activity. Behaviorally, face masks increased listening -phonological- errors in speech content retrieval and perceived listening difficulty. At the neural level, we observed that the occlusion of the mouth abolished lip tracking and dampened neural tracking of the speech envelope at the earliest processing stages. Degraded acoustic information due to face mask filtering altered neural tracking at later processing stages instead. Finally, a consistent link emerged between the increment of listening perceived difficulty and the drop in reconstruction performance of speech envelope when attending to a speaker wearing a face mask. Results clearly dissociated the visual and auditory impacts of face masks on face-to-face neural tracking of speech. While face masks hampered the ability to predict and integrate audio-visual speech, the auditory filter generated by face masks impacted the neural processing stages typically associated with auditory selective attention. The link between perceived difficulty and neural tracking drop provided evidence of a major impact of face masks on the metacognitive levels subtending speech processing.

Intercalated amygdala dysfunction drives extinction deficits in the Sapap3 mouse model of obsessive-compulsive disorder
Background The avoidance of aversive stimuli due to negative reinforcement learning is critical for survival in real-world environments, which demand dynamic responding to both positive and negative stimuli that often conflict with each other. Individuals with obsessive-compulsive disorder (OCD) commonly exhibit impaired negative reinforcement and extinction, perhaps involving deficits in amygdala functioning. An amygdala subregion of particular interest is the intercalated nuclei of the amygdala (ITC) which has been linked to negative reinforcement and extinction, with distinct clusters mediating separate aspects of behavior. This study focuses on the dorsal ITC cluster (ITCd) and its role in negative reinforcement during a complex behavior that models real-world dynamic decision making. Methods We investigated the impact of ITCd function on negative reinforcement and extinction by applying fiber photometry measurement of GCamp6f signals and optogenetic manipulations during a platform-mediated avoidance task in a mouse model of OCD-like behavior: the Sapap3-null mouse. Results We find impaired neural activity in the ITCd of male and female Sapap3-null mice to the encoding of negative stimuli during platform-mediated avoidance. Sapap3-null mice also exhibit deficits in extinction of avoidant behavior, which is modulated by ITCd neural activity. Conclusions Sapap3-null mice fail to extinguish avoidant behavior in platform-mediated avoidance, due to heightened ITCd activity. This deficit can be rescued by optogenetically inhibiting ITCd during extinction. Together, our results provide insight into the neural mechanisms underpinning negative reinforcement deficits in the context of OCD, emphasizing the necessity of ITCd in responding to negative stimuli in complex environments.

A latent pool of neurons silenced by sensory-evoked inhibition can be recruited to enhance perception
Which patterns of neural activity in sensory cortex are relevant for perceptual decision-making? To address this question, we used simultaneous two-photon calcium imaging and targeted two-photon optogenetics to probe barrel cortex activity during a perceptual discrimination task. Head-fixed mice discriminated bilateral whisker deflections and reported decisions by licking left or right. Two-photon calcium imaging revealed sparse coding of contralateral and ipsilateral whisker input in layer 2/3 while most neurons did not show task-related activity. Activating small groups of pyramidal neurons using two-photon holographic photostimulation evoked a perceptual bias that scaled with the number of neurons photostimulated. This effect was dominated by the optogenetic activation of a small number of non-coding neurons, which did not show sensory or motor-related activity during task performance. Patterned photostimulation also revealed potent recruitment of cortical inhibition during sensory processing, which strongly and preferentially suppressed non-coding neurons. Our results provide a novel perspective on the circuit basis for the sparse coding model of somatosensory processing in which a pool of non-coding neurons, selectively suppressed by strong network inhibition during whisker stimulation, can be recruited to enhance perception.

Zapit: Open Source Random-Access Photostimulation For Neuroscience
Optogenetic tools are indispensable for understanding the causal neural mechanisms underlying animal behaviour. While optogenetic actuators provide millisecond-precision control over genetically defined neural populations, successful optogenetic experiments also critically depend on associated hardware for targeted light delivery. Optic-fibres are suitable for certain experiments, however fibre implantation can be invasive and limits flexibility of spatial targeting. In contrast, random-access laser-scanning optogenetic systems provide far greater flexibility for targeting distributed cortical areas. However, these systems can be technically challenging to build, and at present no open source solution is available. Here we present "Zapit", a complete open source platform for spatio-temporally precise random-access laser-scanning optogenetic experiments in head-fixed mice. We describe the system, quantify its performance, and show results from proof of principle cortical photoinhibition experiments in behaving mice.

Age-dependent dynamics of neuronal VAPBALS inclusions in the adult brain
Amyotrophic Lateral Sclerosis (ALS) is a relentlessly progressive and fatal disease caused by the degeneration of upper and lower motor neurons within the brain and spinal cord in the ageing human. The dying neurons contain cytoplasmic inclusions linked to the onset and progression of the disease. Here, we use a Drosophila model of ALS8 (VAPP58S) to understand the modulation of these inclusions in the ageing adult brain. The adult VAPP58S fly shows progressive deterioration in motor function till its demise 25 days post-eclosion. The density of VAPP58S-positive brain inclusions is stable for 5-15 days of age. In contrast, adding a single copy of VAPWT to the VAPP58S animal leads to a large decrease in inclusion density with concomitant rescue of motor function and lifespan. ER stress, a contributing factor in disease, shows reduction with ageing for the disease model. Autophagy, rather than the Ubiquitin Proteasome system, is the dominant mechanism for aggregate clearance. We explored the ability of Drosophila Valosin-containing protein (VCP/TER94), the ALS14 locus, which is involved in cellular protein clearance, to regulate age-dependent aggregation. Contrary to expectation, TER94 overexpression increased VAPP58S punctae density, while its knockdown led to enhanced clearance. Expression of a dominant positive allele, TER94R152H, further stabilised VAPP58S puncta, cementing roles for an ALS8-ALS14 axis. Our results are explained by a mechanism where autophagy is modulated by TER94 knockdown. Our study sheds light on the complex regulatory events involved in the neuronal maintenance of ALS8 aggregates, suggesting a context-dependent switch between proteasomal and autophagy-based mechanisms as the larvae develop into an adult. A deeper understanding of the nucleation and clearance of the inclusions, which affect cellular stress and function, is essential for understanding the initiation and progression of ALS.

Psilocybin induces dose-dependent changes in functional network organization in rat cortex
Psilocybin produces an altered state of consciousness in humans and is associated with complex spatiotemporal changes in brain networks. Given the emphasis on rodent models for mechanistic studies, there is a need for characterization of the effect of psilocybin on brain-wide network dynamics. Previous rodent studies of psychedelics, using electroencephalogram, have primarily been done with sparse electrode arrays that offered limited spatial resolution precluding network level analysis, and have been restricted to lower gamma frequencies. Therefore, in the study, we used electroencephalographic recordings from 27 sites (electrodes) across rat cortex (n=6 male, 6 female) to characterize the effect of psilocybin (0.1 mg/kg, 1 mg/kg, and 10 mg/kg delivered over an hour) on network organization as inferred through changes in node degree (index of network density) and connection strength (weighted phase-lag index). The removal of aperiodic component from the electroencephalogram localized the primary oscillatory changes to theta (4-10 Hz), medium gamma (70-110 Hz), and high gamma (110-150 Hz) bands, which were used for the network analysis. Additionally, we determined the concurrent changes in theta-gamma phase-amplitude coupling. We report that psilocybin, in a dose-dependent manner, 1) disrupted theta-gamma coupling [p<0.05], 2) increased frontal high gamma connectivity [p<0.05] and posterior theta connectivity [p less than or equal to 0.049], and 3) increased frontal high gamma [p<0.05] and posterior theta [p less than or equal to 0.046] network density. The medium gamma frontoparietal connectivity showed a nonlinear relationship with psilocybin dose. Our results suggest that high-frequency network organization, decoupled from local theta-phase, may be an important signature of psilocybin-induced non-ordinary state of consciousness.