2025-04-10 | | Total: 26
Prenatal nicotine exposure impairs fetal cortical grey matter volume, but the precise cellular mechanisms remain poorly understood. This study elucidates the role of nicotinic acetylcholine receptors (nAChRs) in progenitor cells and radial glia (RG) during human cortical development. We identify two nAChR subunits, CHRNA7 and the human-specific CHRFAM7A expressed in SOX2+ progenitors and neurons, with CHRFAM7A particularly enriched along RG endfeet. nAChR activation in organotypic slices and dissociated cultures increases RG proliferation while decreasing neuronal differentiation, whereas nAChR knockdown reduces RG and increases neurons. Single-cell RNA sequencing reveals that nicotine exposure downregulates key genes in excitatory neurons (ENs), with CHRNA7 or CHRFAM7A selectively modulating these changes, suggesting an evolutionary divergence in regulatory pathways. Furthermore, we identify YAP1 as a critical downstream effector of nAChR signaling, and inhibiting YAP1 reverses nicotine-induced phenotypic alterations in oRG cells, highlighting its role in nicotine-induced neurodevelopmental pathophysiology.
Glycogen is the largest energy reserve in the brain, but the specific role of glycogen in supporting neuronal energy metabolism in vivo is not well understood. We established a system in C. elegans to dynamically probe glycolytic states in single cells of living animals via the use of the glycolytic sensor HYlight and determined that neurons can dynamically regulate glycolysis in response to activity or transient hypoxia. We performed an RNAi screen and identified that PYGL-1, an ortholog of the human glycogen phosphorylase, is required in neurons for glycolytic plasticity. We determined that neurons employ at least two mechanisms of glycolytic plasticity: glycogen-dependent glycolytic plasticity (GDGP) and glycogen-independent glycolytic plasticity (GIGP). We uncover that GDGP is employed under conditions of mitochondrial dysfunction, such as transient hypoxia or in mutants for mitochondrial function. We find that the ability of neurons to plastically regulate glycolysis through cell-autonomous GDGP is important for sustaining the synaptic vesicle cycle. Together, our study reveals that, in vivo, neurons can directly use glycogen as a fuel source to sustain glycolytic plasticity and synaptic function.
The integrity and function of the blood-brain barrier (BBB) are largely regulated by pericytes. Pericyte deficiency leads to BBB breakdown and neurological dysfunction in major neurological disorders including stroke and Alzheimer's disease (AD). Transplantation of pericytes derived from induced pluripotent stem cells (iPSC-PC) has been shown to restore the BBB and improve functional recovery in mouse models of stroke and pericyte deficiency. However, the molecular profile and functional properties of iPSC-PC under hypoxic conditions, similar to those found in ischemic and neurodegenerative diseases remain largely unexplored. Here, we demonstrate that iPSC-PC under severe hypoxia retain essential functional properties, including key molecular markers, proliferation rates, and the ability to migrate to host brain vessels via function-associated PDGFRB-PDGFBB signaling. Additionally, we show that iPSC-PC exhibit similar clearance of amyloid betaneurotoxins from AD mouse brain sections under both normoxic and hypoxic conditions. These findings suggest that iPSC-PC functions are largely resilient to hypoxia, highlighting their potential as a promising cell source for treating ischemic and neurodegenerative disorders.
Humans excel at adjusting movements and acquiring new skills through feedback corrections and predictive control, yet how these feedback-feedforward computations evolve in the motor system remains unclear. We investigated this process by examining how humans learned a novel, continuous visuomotor mirror reversal (MR) tracking task over multiple days. Using a frequency-dependent system-identification approach and responses to cursor perturbations, we dissociated feedback-driven corrections from predictive feedforward adjustments. Our findings reveal two distinct learning pathways: early learning relies on rapid, corrective feedback at lower frequencies, while feedforward control gradually emerges at higher frequencies, compensating for feedback limitations. These findings suggest that motor learning involves a dynamic interplay between feedback and feedforward control, providing mechanistic insights into sensorimotor learning, with implications for optimizing motor skill acquisition and neurorehabilitation strategies
Animals continuously combine information across sensory modalities and time, and use these combined signals to guide their behaviour. Picture a predator watching their prey sprint and screech through a field. To date, a range of multisensory algorithms have been proposed to model this process including linear and nonlinear fusion, which combine the inputs from multiple sensory channels via either a sum or nonlinear function. However, many multisensory algorithms treat successive observations independently, and so cannot leverage the temporal structure inherent to naturalistic stimuli. To investigate this, we introduce a novel multisensory task in which we provide the same number of task-relevant signals per trial but vary how this information is presented: from many short bursts to a few long sequences. We demonstrate that multisensory algorithms that treat different time steps as independent, perform sub-optimally on this task. However, simply augmenting these algorithms to integrate across sensory channels and short temporal windows allows them to perform surprisingly well, and comparably to fully recurrent neural networks. Overall, our work: highlights the benefits of fusing multisensory information across channels and time, shows that small increases in circuit/model complexity can lead to significant gains in performance, and provides a novel multisensory task for testing the relevance of this in biological systems. Key Points We introduce a novel multisensory task in which we provide task relevant evidence via bursts of varying duration, amidst a noisy background. Prior multisensory algorithms perform sub-optimally on this task, as they cannot leverage temporal structure. However, they can perform better by integrating across sensory channels and short temporal windows. Surprisingly, this allows for comparable performance to fully recurrent neural networks, while using less than one tenth the number of parameters.
Exposure to stress can increase the risk of depression in susceptible individuals, but not in resilient ones. Resilience to stress decreases with age, parallel to drastic changes in the quality of brain plasticity from juvenile to old age, suggesting that the type of plasticity found in the maturing brain promotes resilience. To indirectly test this, we administered short unpredictable stress to adult male and female mice, as well as to adolescent mice and mice that lack SynCAM 1 and display extended adolescent-like, critical period plasticity in the brain. We found that short unpredictable stress robustly increased core body temperature in all groups of mice, indicative of stress-induced hyperthermia (SIH) and confirming the efficacy of the stress paradigm. However, depressive-like behavior as measured though tail suspension test was increased in adult mice only, supporting that the type of plasticity found in the maturing brain promotes resilience to stress. All three groups of mice showed a mild increase in locomotor activity after stress, suggesting that the quality of plasticity does not correlate with resilience to anxiety-like phenotypes. Our study hence provides further evidence for the protective role of developmental plasticity during stress and points to new mechanisms that promote resilience to stress-induced depression.
During vocalization, mismatches between expected and perceived auditory feedback are processed rapidly and automatically, suggesting that feedback control of vocalization operates unconsciously. However, whether consciousness modulates speech feedback control remains little studied. To address this question, we concurrently measured behavioral vocal responses and electroencephalography (EEG) in 30 participants while they vocalized and their auditory feedback was perturbed with individually calibrated perceptual threshold level pitch shifts. Following each vocalization trial, participants rated if they consciously detected a pitch shift in their auditory feedback. We analyzed the data on a trial-by-trial basis to test if vocal responses to pitch perturbations were modulated by conscious perception. Our results revealed that even on trials where the participants reported not noticing the pitch shift at all, a compensatory vocal response to the altered auditory feedback was generated. Conscious detection of a pitch shift was associated with an increased magnitude of vocal responses roughly 500-700 ms after the pitch shift compared to the unconscious trials. Conscious detection of the pitch shift correlated with early (Auditory awareness negativity, AAN) and late (Late positivity, LP) neural responses as indexed by the modulation of event-related potentials (ERPs). Source localization of the ERPs suggested that conscious pitch shift detection was associated with increased neural activity within the temporal, frontal and parietal cortical networks known to be involved in speech motor control. These findings emphasize the importance of investigating the role of consciousness in regulating speech feedback control, and their effect on the underlying neural and behavioral functions.
Descending neurons (DNs) occupy a key position in the sensorimotor hierarchy, conveying signals from the brain to the rest of the body below the neck. In Drosophila melanogaster flies, approximately 480 DN cell types have been described from electron-microscopy image datasets. Genetic access to these cell types is crucial for further investigation of their role in generating behaviour. We previously conducted the first large-scale survey of Drosophila melanogaster DNs, describing 98 unique cell types from light microscopy and generating cell-type-specific split-Gal4 driver lines for 65 of them. Here, we extend our previous work, describing the morphology of 137 additional DN types from light microscopy, bringing the total number DN types identified in light microscopy datasets to 235, or nearly 50%. In addition, we produced 500 new sparse split-Gal4 driver lines and compiled a list of previously published DN lines from the literature for a combined list of 738 split-Gal4 driver lines targeting 171 DN types.
Learned information and experiences are thought to be stored in synapses, composed of building block molecules whose number typically correlates with synaptic strength. Activity-dependent plasticity mechanisms, such as Hebbian learning, regulate these building blocks, promoting synaptic growth to encode acquired knowledge. However, this process can destabilize cortical networks through overexcitation, leading to runaway dynamics. To prevent such instabilities the brain uses compensatory mechanisms like synaptic scaling. Existing models rely on rapid timescales, contradicting experimental observations that synaptic scaling occurs slowly. Here, we introduce aggregate scaling, a simple framework to study synapse-mediated homeostasis based on the availability and competitive redistribution of synaptic building blocks. Our model enforces stability by integrating rapid regulation of the total synaptic strength and firing rate homeostasis over much slower, realistic timescales. It preserves key neuronal properties, such as firing activity around a homeostatic set-point, long-tailed distributions of synaptic weights, and response to brief stimulation.
Cajal-Retzius (CR) cells are glutamatergic neurons that transiently populate the most superficial layer of the isocortex and allocortex during development, serving an essential role during both prenatal and early postnatal brain development. Notably, these cells disappear from most cortical areas by postnatal day 14, but persist for much longer in the hippocampus. We developed a novel intersectional genetic labeling approach for CR cells that captures almost all of the TRP73-positive CR cells throughout the isocortex and allocortex. This intersectional strategy offers several advantages over previous methods commonly used for CR cell targeting. Here, we applied this new CR cell labeling strategy to investigate the distribution and persistence of CR cells throughout the whole mouse brain, at four different postnatal ages. We observed that the initial CR cell density and the rate of their disappearance varies substantially across different brain areas during development. Strikingly, we observed variation in cell death rate even between adjacent cortical subregions: comparing the medial and the lateral entorhinal cortex, the former retains a high density of CR cells for several months in contrast to the latter. Our results present a necessary revision of the phenomenon of CR cell persistence, showing that, in addition to hippocampus, several other cortical areas maintain a high density of these cells beyond the first two postnatal weeks.
Disorders characterised by changes in dopamine (DA) neurotransmission are often linked to changes in the temporal discounting of future rewards. Likewise, pharmacological manipulations of DA neurotransmission in healthy individuals modulates temporal discounting, but there is considerable variability in the directionality of reported pharmacological effects, as enhancements and reductions of DA signalling have been linked to both increases and reductions of temporal discounting. This may be due to meaningful individual differences in drug effects and/or false positive findings in small samples. To resolve these inconsistencies, we 1) revisited pharmacological effects of the DA precursor L-DOPA on temporal discounting in a large sample of N = 76 healthy participants (n = 44 male) and 2) examined several putative proxy measures for DA to revisit the role of individual differences in a randomised, double-blind placebo-controlled pre-registered study (https://osf.io/a4k9j/). Replicating previous findings, higher rewards were discounted less (magnitude effect). Computational modelling using hierarchical Bayesian estimation confirmed that the data in both drug conditions were best accounted for by a non-linear temporal discounting drift diffusion model. In line with recent animal and human work, L-DOPA reliably reduced the discount rate with a small effect size, challenging earlier findings in substantially smaller samples. We found no credible evidence for linear or quadratic effects of putative DA proxy measures on model parameters, calling into question the role of these measures in accounting for individual differences in DA drug effects.
Pharmacologic lowering of PrP expression is efficacious against prion disease in animal models and is now being tested clinically. 50% lowering of PrP increases both survival time and healthy life in prion-infected mice, but does not prevent symptom onset nor halt disease progression. Additional drug candidates should seek to reduce PrP expression to even lower levels. Divalent siRNA is a novel oligonucleotide drug modality with promising potency, durability, and biodistribution data in preclinical models, inspiring us to seek in this technology a new drug candidate for prion disease. Here, we first identify a tool compound against the mouse PrP gene and establish the efficacy of PrP-lowering divalent siRNA in prion-infected mice. We then introduce humanized transgenic mouse lines harboring the full non-coding sequence of the human PrP gene as tools for identifying human sequence-targeted drugs. We identify a highly potent siRNA sequence against the human PrP gene and determine that a chemical scaffold incorporating extended nucleic acid and a 3′ antisense tail unmatched to the RNA target yields superior potency. We nominate PrP-lowering divalent siRNA 2439-s4 as a new drug candidate for human prion disease.
The lateral hypothalamic area (LHA) contains GABAergic and glutamatergic neurons that converge on the midbrain dopamine system and exert opposing influences on consummatory feeding behavior. However, the activity dynamics of these populations during consumption and their impact on striatal dopamine release remain poorly understood. Here, we show that LHA GABAergic and glutamatergic neurons independently scale their activity during the consumption of rewarding and aversive solutions while cooperatively regulating dopamine release along the anterior-posterior axis of the striatum in mice. Dopamine release exhibited widespread modulation during consumption, with anterior striatal regions showing stronger representation of solution value and its recent history - an effect dependent on LHA activity. These findings suggest that the LHA acts as a central regulator of striatal dopamine release during consumption, providing a mechanism through which hypothalamic circuits influence motivational and consummatory behaviors.
Auditory cortex possesses a remarkable ability to discriminate between tone sequences with different levels of statistical regularities. To examine this phenomenon at the level of single neurons, we recorded responses from auditory cortex of awake rats. The rats passively listened to multitone sequences composed of 4, 5 or 6 tones. The tones could be presented in periodic sequences with a fixed, repeated cycle; in a fully random condition; or in an intermediate condition in which cycles were maintained but tones were randomly permuted within each cycle (random cycle condition). Units showed a continuum of preferences between order-preferring neurons and randomness-preferring neurons. Unexpectedly, we found sensitivity to the position of sounds in the cycle ('phase modulation'). The strongest and most consistent phase modulations were in the responses to the random cycle condition. Finally, we show how such sensitivities may emerge from a simple, biologically-plausible computation.
Atypical sensory processing in neurodevelopmental disorders contributes to cognitive, social, and behavioural disruptions, yet underlying neurophysiological mechanisms remain unclear. Using a mouse model of SYNGAP1 haploinsufficiency (HET), a common monogenic cause of intellectual disability and autism, we investigated visual processing deficits. Syngap HET mice exhibited impaired behavioural visual discriminability, associated with reduced coding precision for visual stimuli in the primary visual cortex (V1). Notably, intrinsic properties of V1 neurons and visual responses under anaesthesia were unaltered, suggesting behavioural state-dependent disruptions in awake Syngap HET mice. Supporting this, both mice and individuals with SYNGAP1 haploinsufficiency exhibited larger pupil size during visual stimulation, implicating neuromodulatory dysfunction. Targeting noradrenergic tone systemically with an α2-adrenergic receptor agonist restored V1 coding precision in Syngap HET mice. Our findings reveal neuromodulatory dysregulation as a novel mechanism underlying sensory disruptions in SYNGAP1-related disorder, highlighting potential therapeutic targets for addressing sensory impairments in neurodevelopmental disorders.
Our brain constantly forms new memories and stabilizes existing memories. To achieve such cognitive flexibility, the brain is wired by plastic synapses that are hotspots of energy consumption. Supplying energy to distant synapses is challenging as they are distributed throughout dendrites and axons, spanning hundreds of microns from their cell body. Synapses, therefore, require an instant and local energy supply provided by mitochondria stabilized near dendritic spines. However, the mechanisms by which synapses communicate their energy demands to locally stable mitochondria to drive local energy production and sustain synaptic plasticity is unknown. Using highly sensitive spine- and mitochondrial ATP reporters and two-photon glutamate uncaging to stimulate individual spines, we find that synaptic plasticity input drives instant and sustained increase in spine ATP levels, provided by local ATP synthesis in ~10-20 μm spatially confined compartments within mitochondria. This spatially localized mitochondrial ATP generation is driven by a spatially localized mitochondrial calcium influx independent of the endoplasmic reticulum. Notably, the initial spine ATP increase, supported by local mitochondrial ATP synthesis, is independent of CaMKII and the energy demands of spine structural plasticity. Without local calcium signaling and mitochondrial stabilization, synapses do not meet their instant and sustained energy needs, resulting in synaptic plasticity defects, as observed in neurological disorders.
The attentional blink reflects a ubiquitous bottleneck with selecting and processing the second of two targets that occur in close temporal proximity. An extensive literature has examined the attention blink as a unitary phenomenon, As a result, which specific component of attention – perceptual sensitivity or choice bias – is compromised during the attentional blink, and their respective neural bases, remains unknown. Here, we address this question with a multialternative task and novel signal detection model, which decouples sensitivity from bias effects. We find that the attentional blink impairs specifically one component of attention – sensitivity – while leaving the other component – bias – unaffected. Distinct neural markers of the attentional blink mapped on to distinct subcomponents of the sensitivity deficits. Parieto-occipital N2p and P3 potential amplitudes characterized target detection deficits whereas long-range high-beta band (20-30 Hz) coherence between frontoparietal electrodes signalled target discrimination deficits. We synthesized these results with representational geometry analysis. The analysis revealed that detection and discrimination deficits were encoded along separable neural dimensions, whose configural distances robustly correlated with the neural markers of each. Overall, these findings shed new light on subcomponents of the attentional blink, and reveal dissociable neural bases underlying its detection and discrimination bottlenecks. Lay summary In daily life, our attention switches rapidly between different objects. For example, when driving, we may shift focus from a billboard on the roadside to a pedestrian in front, in quick succession. Yet, our ability to process the second object is severely compromised especially when it appears soon after the first: an impairment called the “attentional blink”. In previous work, the attentional blink has been studied essentially as a “monolithic” (indivisible) phenomenon. We design a behavioral model to divide the attentional blink into sub-components and show that the blink affects only one specific component (sensitivity). We also identify key neural markers for deficits associated with this component. Our findings may aid in understanding the neural origins of attention deficit disorders.
In the developing cerebral cortex astrocytes arise from progenitors in the ventricular and subventricular zones (V-SVZ), and also from local proliferation within the parenchyma. In the mouse neocortex, astrocytes that occupy upper versus deep layers (UL/DL) are known to be distinct populations in terms of molecular and morphological features. Transcription factor LHX2 is expressed both in V-SVZ gliogenic progenitors and in differentiated astrocytes throughout development and into adulthood. Here we show that loss of Lhx2 at birth results in an increased astrocyte proliferation in UL but not the DL of the cortex in the first postnatal week. Consistent with this, transcriptomic signatures of UL astrocytes increase. By 3 months, Lhx2 mutant astrocytes display upregulation of GFAP, and transcriptomic signatures associated with reactive astrocytes, in the absence of injury. These results demonstrate a novel role for Lhx2 in regulating proliferation and molecular features of cortical astrocytes.
The hypothalamus is crucial for regulating essential bodily functions, including energy balance. It is an exceedingly complex and heterogeneous brain region that contains a variety of neuronal systems that are interconnected with each other. Among these, the melanocortin system, which comprises pro-opiomelanocortin (POMC) and agouti-related peptide (AgRP) neurons, displays a remarkable anatomical relationship with oxytocin (OT) neurons in the paraventricular nucleus (PVH). Here, we demonstrate that OT neurons are instrumental in the development of the melanocortin system. Chemogenetic inhibition of OT neurons during the first postnatal week selectively disrupts POMC and AgRP projections to the PVH, without affecting other target nuclei like the dorsomedial nucleus. This developmental role is age-dependent, as silencing OT neurons in juvenile or adult stages has no impact on melanocortin circuits. OT neurons release various neuropeptides and neurotransmitters, and their secretion can be modulated by chemogenetic manipulation. Expressing the botulinum toxin serotype B light chain in OT neurons reveals that their developmental actions rely on SNARE-mediated exocytosis. Moreover, administering an OT receptor antagonist during the first postnatal week leads to similar melanocortin circuit defects and long-term metabolic effects. Furthermore, neonatal chemogenetic activation of OT neurons rescues POMC circuit deficits in a mouse model of Prader-Willi Syndrome. These findings reveal that OT acts as a paracrine neurotrophic factor orchestrating the development of melanocortin circuits during a restricted neonatal critical period.
A salient visual object with a distinct feature from the surrounding environment automatically captures attention. While the saliency signals have been found in many brain regions, their source remains highly controversial. Here, we investigated the neural origin of visual saliency using cortical layer-dependent functional magnetic resonance imaging (fMRI) of cerebral blood volume (CBV) at 7 Tesla. Behaviorally, human observers were better at detecting foreground bars with a larger orientation contrast from uniformly oriented background bars. Saliency-sensitive signals were strongest in the superficial layers of the primary visual cortex (V1), and in the middle layers of the intraparietal sulcus (IPS) of the parietal cortex. Layer-dependent effective connectivity revealed the transmission of saliency signals along the feedforward pathway from V1 to IPS. Furthermore, behavioral sensitivity to the foreground stimulus correlated significantly with the fMRI response in the superficial layers of V1. Our findings provide mesoscale evidence that a visual saliency map is created by iso-feature suppression through lateral inhibition in the superficial layers of V1, and then feeds forward to attentional control brain regions to guide attention and eye movements.
Citation metrics influence academic reputation and career trajectories. Recent works have highlighted flaws in citation practices in the Neurosciences, such as the under-citation of women. However, self-citation rates—or how much authors cite themselves—have not yet been comprehensively investigated in the Neurosciences. This work characterizes self-citation rates in basic, translational, and clinical Neuroscience literature by collating 100,347 articles from 63 journals between the years 2000-2020. In analyzing over five million citations, we demonstrate four key findings: 1) increasing self-citation rates of Last Authors relative to First Authors, 2) lower self-citation rates in low- and middle-income countries, 3) gender differences in self-citation stemming from differences in the number of previously published papers, and 4) variations in self-citation rates by field. Our characterization of self-citation provides insight into citation practices that shape the perceived influence of authors in the Neurosciences, which in turn may impact what type of scientific research is done and who gets the opportunity to do it.
A central challenge in Alzheimers disease is understanding the mechanism of neuronal secretory dysfunction. By exploiting AI, we reveal how beta-amyloid disrupts key protein interactions within the neuronal secretory machinery. In a combinatorial strategy of reprogramming the neuronal secretory and metabolic components, we established a dual-target therapeutic framework that repairs synaptic and mitochondrial defects to counteract neurodegeneration in Alzheimers.
Intestinal infections trigger inflammation and can contribute to degenerative and cognitive brain pathologies through the microbiota-gut-brain axis. Galectin-4 is an intestinal lectin key in the control of pathogenic bacterial infections. Here we report that galectin-4 deficient mice (Lgals4-KO) show an altered intestinal commensal microbiota in the absence of pathogens, defining a new role of galectin-4 in the modulation of commensal bacteria. Strikingly, Lgals4-KO mice present a deficient memory formation, and impaired hippocampal long-term potentiation (LTP) in vivo and ex vivo. Furthermore, Lgals4-KO neurons show a reduced activation of the AMPA receptor and of the CaMKII upon chemically induced LTP in vitro. These mice also display significantly lower dendritic spine density and shorter spine length in hippocampal dendrites, as well as an increased area of the postsynaptic densities, all coherent with an alteration of the synaptic function. In all, our results demonstrate that the absence of galectin-4 induces synaptic dysfunctions and memory impairment, along with changes in gut microbial composition, suggesting that variations in endogenous intestinal microbiota may cause or contribute to such neurological pathologies.
Patients with cerebellar damage experience various motor impairments, but the specific sequence of primary and compensatory processes that contribute to these deficits remains uncertain. To clarify this, we reversibly blocked cerebellar outflow in monkeys engaged in planar reaching tasks. This intervention led to a spatially selective reduction in hand velocity, primarily due to decreased muscle torque, especially in movements requiring high inter-joint torque coupling. When examining repeated reaches to the same target, we found that the reduced velocity resulted from both an immediate deficit and a gradually developing compensatory slowing strategy designed to reduce passive inter-joint interactions. However, the slowed hand velocity did not account for the fragmented and variable movement trajectories observed during the cerebellar block. Our findings indicate that cerebellar impairment results in motor deficits due to both inadequate muscle torque and an altered compensatory control strategy for managing impaired limb dynamics. Additionally, impaired feedforward control elevates motor noise, which cannot be entirely mitigated through compensatory strategies.
Regulated secretion typically depends on activity-induced Ca2+ influx. However, in invertebrates, the endoplasmic reticulum (ER) plays a distinct role, particularly in the release of neuromodulators from dense-core vesicles (DCVs). Here, we investigated the role of the neuronal ER as a Ca2+ source for neuromodulator secretion in primary mouse neurons by directly monitoring ER and cytosolic Ca2+ dynamics, along with DCV exocytosis at single vesicle resolution. During neuronal activity, neurons with a low initial [Ca2+]ER took up Ca2+ into the ER, while those with a high initial [Ca2+]ER released ER Ca2+. These latter neurons showed more DCV exocytosis. Acute ER Ca2+ release by caffeine or thapsigargin application, resulted in minute increases in bulk cytosolic free Ca2+ that did not trigger DCV exocytosis. Remarkably, following ER Ca2+ depletion levels, activity-dependent Ca2+ influx and DCV exocytosis were reduced by 50-90%, while synaptic vesicle (SV) exocytosis was unaffected. L-type Ca2+-channel inhibition by nimodipine reduced DCV exocytosis and Ca2+ influx by 80-90 % without affecting SV exocytosis, a phenocopy of ER store depletion. In addition, introducing L-type channels lacking STIM1 interaction sites restored DCV fusion following ER store depletion. We conclude that the ER functions as a dynamic Ca2+ store serving both as a Ca2+ source or sink. Moreover, ER depletion activates a feedback loop that controls L-type Ca2+ channel activity, essential for DCV exocytosis.